KR20240036634A - Peptide compositions capable of binding to lanthionine synthase C-like protein (LANCL) and uses thereof - Google Patents

Abstract

The present invention provides various peptide compositions capable of binding to lanthionine synthase C-like (LanCL) protein, which has analgesic, anti-inflammatory and antimicrobial properties. _{The composition} comprises _a peptide _of formula ( _{I )} : It is not a linear peptide containing the sequence EQLERALNSS and does not contain CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC.

Description

Peptide compositions capable of binding to lanthionine synthase C-like protein (LANCL) and uses thereof

The present invention generally relates to peptides and their uses suitable for treating conditions such as pain, inflammatory conditions and respiratory infections.

All references, including any patents or patent applications, cited herein are incorporated by reference to enable a complete understanding of the invention. Nevertheless, such references should not be construed as an admission that any of these documents forms part of the common general knowledge in the art in Australia or any other country.

As pointed out by Lee et al. (Int J Mol Sci. 2019; 20(10): 2383), Protein-protein interactions (PPIs) are the basis of almost all cellular processes. These biochemical processes often consist of activated receptors that indirectly or directly regulate a series of cell signaling events that regulate transcription of nucleic acids and/or post-translational modifications of translated proteins. Drugs that bind specifically to such receptors can act as agonists or antagonists, which have subsequent effects on cell behavior. Therefore, peptides and small molecules that interfere with PPIs are attracting attention as therapeutic agents due to their potential to modulate disease-related protein interactions. The authors note that better identification of targetable, disease-relevant PPIs and optimization of peptide drug-binding properties will be key to its clinical success. However, understanding the molecular recognition mechanism and elucidating the binding affinity for PPIs is a complex challenge for both computational biologists and protein biochemists. The main reason is that small molecules are superior at binding to deep folded protein pockets compared to the larger, flatter, and hydrophobic binding interfaces commonly present at PPI complex interfaces. Antibodies are generally more effective at recognizing these PPI interfaces, but are generally unable to penetrate the cell membrane to reach and recognize intracellular targets. The authors note that peptides with balanced structural flexibility and binding affinity that are up to five times greater than small molecule drugs have recently received considerable attention. For example, cyclic peptides have small molecule drug properties such as robust antibody-like binding affinity and long in vivo stability while maintaining minimal toxicity.

de la Torre and Albericio (2020; Molecules; 25(10): 2293) reported that the field of peptide-based drug discovery has recently shown significant activity, with the US Food and Drug Administration (FDA) approving 208 new drugs from 2015 to 2019. Of these, 150 were new chemical entities and 58 were biological products containing 15 peptides or peptide-containing molecules. These include Ixazomib (N-acylated, C-boronic acid dipeptide for the treatment of multiple myeloma), Adlyxin (34 amino acid analogue of parathyroid hormone-related protein for the treatment of osteoporosis), and Etelcalcetide (disulfide bridge for the treatment of hyperparathyroidism). Ac-DCys-DAla-(DArg) ₃ -DAla-DArg-NH ₂ ) linked to L-Cys via ) and Afamelanotide (a 13-amino acid linear peptide of α-melanocyte-stimulating hormone (αMSH) for the treatment of skin damage and pain. analogues) are included. The authors note that oncology, metabolism, and endocrinology are the most frequent indications for FDA-approved peptide-based therapies, although cardiovascular, gastroenterology, bone disease, dermatology, and sexual dysfunction are also target indications for FDA-approved peptide-based therapies.

Compared to small molecules such as proteins and antibodies, peptides represent a unique class of pharmaceutical compounds due to their distinct biochemical and therapeutic properties. In addition to peptide-based natural hormone analogs, peptides have been developed as drug candidates that disrupt protein-protein interactions (PPIs) and target or inhibit intracellular molecules such as receptor tyrosine kinases. This strategy has transformed peptide therapeutics into a leading industry, with approximately 20 new peptide-based clinical trials each year. In fact, there are currently more than 400 peptide drugs in global clinical development, of which more than 60 have already been approved for clinical use in the United States, Europe, and Japan.

Although peptide-based therapeutics have made significant advances, they have been largely limited to the treatment of specific diseases and conditions corresponding to the PPIs and cell signaling pathways targeted by these peptide-based therapeutics. Accordingly, there remains a continuing need for a broad range of peptide-based therapeutic strategies that can beneficially alleviate a variety of diseases, conditions, or symptoms, including those associated with cellular aging, damage, or stress. The present invention addresses or at least partially alleviates these limitations by providing therapeutic peptides with broad-spectrum activities such as analgesic, anti-inflammatory and antimicrobial activities.

Summary of the Invention

In one aspect disclosed herein, provided is a peptide capable of binding to a lanthionine synthase C-like (LanCL) protein, wherein the peptide comprises an amino acid sequence of Formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (I)

here:

X ₁ is selected from the group consisting of lysine, arginine and histidine, or X ₁ is absent;

X ₂ is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, tyrosine and serine;

X ₃ is selected from the group consisting of glycine, alanine, valine, leucine and isoleucine;

X ₄ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, aspartic acid, lysine, glutamic acid, proline and histidine, or X ₄ is absent;

X ₅ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, lysine, histidine and glycine, or X ₅ is absent;

X ₆ is selected from the group consisting of serine, cysteine, threonine, asparagine, glutamine, tyrosine, and histidine, or X ₆ is absent.

wherein the peptide is 3 to 20 amino acids in length;

wherein the amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC;

wherein the peptide is not a linear peptide comprising the amino acid sequence EQLERALNSS.

In another aspect disclosed herein, provided is a peptide capable of binding to a lanthionine synthase C-like (LanCL) protein, wherein the peptide comprises an amino acid sequence of Formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (I)

here:

X ₁ is selected from the group consisting of lysine, arginine and histidine;

X ₂ is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, tyrosine and serine;

X ₃ is selected from the group consisting of glycine, alanine, valine, leucine and isoleucine;

X ₄ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, aspartic acid, lysine, glutamic acid, proline and histidine, or X ₄ is absent;

X ₅ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, lysine, histidine and glycine, or X ₅ is absent;

X ₆ is selected from the group consisting of serine, cysteine, threonine, asparagine, glutamine, tyrosine, and histidine, or X ₆ is absent.

wherein the peptide is 3 to 20 amino acids in length;

wherein the amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC;

wherein the peptide is not a linear peptide comprising the amino acid sequence EQLERALNSS.

Figures 1A and 1B show the effect of the peptide of SEQ ID NO: 1 on the viability of Taxol-stressed A549 adenocarcinoma human alveolar basal epithelial cells. Cells were treated with LanCL1 siRNA (100 nM for 48 h) to knock down LanCL1 expression. Cells were then incubated in the presence of Taxol (IC _50-350 μM), vehicle alone (dimethylsulfoxide; DMSO), or peptide of SEQ ID NO: 1 (diluted in DMSO) at concentrations of 1, 5, 25, 50, and 100 μM. Y-axis represents relative luminescence units (RLU); The X-axis represents the concentration of peptide.
Figure 2 shows the effect of the peptide of SEQ ID NO: 9 on the viability of Taxol-stressed A549 cells. Cells were treated with Taxol (IC _50-350 μM) in the presence of vehicle alone (DMSO) or peptide of SEQ ID NO:9 (diluted in DMSO) at concentrations of 1, 5, 25, 50, and 100 μM. Y-axis represents relative luminescence units (RLU); The X-axis represents the concentration of peptide.
Figure 3 shows the effect of peptides RSVEGS (SEQ ID NO: 9), SVEGS (SEQ ID NO: 62) and ALNSS (SEQ ID NO: 63) on ipsilateral paw withdrawal threshold (PWT; grams) in the rat Chung model of neuropathic pain. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to vehicle group (one-way ANOVA, n = 6 per group).
Figure 4 shows the effect of peptides RSVEGS (SEQ ID NO: 9), SVEGS (SEQ ID NO: 62) and ALNSS (SEQ ID NO: 63) on contralateral paw withdrawal threshold (PWT; grams) in the rat Chung model of neuropathic pain. ANOVA, n=6 per group).

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention the following terms are defined below.

“One” and “one” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, “element” means one element or more than one element.

As used herein, the term “about” means as much as 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%) of a reference quantity, level, value, dimension, size or amount. means a quantity, level, value, dimension, size or quantity that changes (by %, 3%, 2% or 1%).

Throughout this specification, unless the context otherwise requires, the words "comprise", "comprise" and "comprising" mean including a stated step or element or group of steps or elements, but not other steps. It will also be understood that it does not exclude elements or steps or groups of elements.

peptide

We have previously identified the molecular targets of a new class of cyclic peptide molecules (lanthionine synthase C-like protein, LanCL) that have previously been endowed with analgesic and other therapeutic properties. The work is described in WO2021/127752. We subsequently identified a novel consensus sequence for a peptide (formula ( I)) was confirmed. Moreover, we unexpectedly discovered that many peptides containing this consensus sequence will retain biological activity regardless of whether they are presented in a cyclic or linear peptide configuration. Accordingly, in one aspect disclosed herein, a peptide capable of binding to a lanthionine synthetase C-like (LanCL) protein is provided, wherein the peptide comprises an amino acid sequence of Formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (I)

here:

X ₁ is selected from the group consisting of lysine, arginine and histidine;

X ₂ is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, tyrosine and serine;

X ₃ is selected from the group consisting of glycine, alanine, valine, leucine and isoleucine;

X ₄ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, aspartic acid, lysine, glutamic acid, proline and histidine, or X ₄ is absent;

X ₅ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, lysine, histidine and glycine, or X ₅ is absent;

X ₆ is selected from the group consisting of serine, cysteine, threonine, asparagine, glutamine, tyrosine, and histidine, or X ₆ is absent.

wherein the peptide is 3 to 20 amino acids in length;

wherein the amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC;

wherein the peptide is not a linear peptide comprising the amino acid sequence EQLERALNSS.

In one embodiment, X ₁ is arginine. In one embodiment, the peptide is not a linear peptide comprising the amino acid sequence QEQLERALNSS.

We have also unexpectedly shown that the peptides described herein exhibit at least some of the biological activities previously attributed to this class _of cyclic LanCL-binding peptides, including analgesic, anti-inflammatory and antimicrobial activities, even in the absence of unexpectedly showed that it possesses Accordingly, in the embodiments described herein, X ₁ is absent.

In another aspect disclosed herein, a peptide capable of binding to a lanthionine synthase C-like (LanCL) protein is provided, wherein the peptide comprises an amino acid sequence of Formula (I):

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (I)

here:

X ₁ is selected from the group consisting of lysine, arginine and histidine, or X ₁ is absent;

X ₂ is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, tyrosine and serine;

X ₃ is selected from the group consisting of glycine, alanine, valine, leucine and isoleucine;

X ₄ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, aspartic acid, lysine, glutamic acid, proline and histidine, or X ₄ is absent;

X ₅ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, lysine, histidine and glycine, or X ₅ is absent;

X ₆ is selected from the group consisting of serine, cysteine, threonine, asparagine, glutamine, tyrosine, and histidine, or X ₆ is absent.

wherein the peptide is 3 to 20 amino acids in length;

wherein the amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC;

wherein the peptide is not a linear peptide comprising the amino acid sequence EQLERALNSS.

In one embodiment, the peptide is not a linear peptide comprising the amino acid sequence QEQLERALNSS.

In one embodiment, the amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, CRIIHNNNC, CRRFVESSCA, or CRIVYDSNC.

In one embodiment, X ₂ is selected from the group consisting of alanine, isoleucine, proline, phenylalanine, and serine.

In one embodiment, X ₃ is selected from the group consisting of valine, leucine, and isoleucine.

In one embodiment, X ₄ is selected from the group consisting of asparagine, glutamic acid, and histidine, or X ₄ is absent. In one embodiment, X ₄ is selected from the group consisting of asparagine, glutamic acid, proline, and histidine. In one embodiment, X ₄ is absent.

In one embodiment, X ₅ is selected from the group consisting of serine, asparagine, and glycine, or X ₅ is absent. In one embodiment, X ₅ is selected from the group consisting of serine, asparagine, and glycine. In one embodiment, X ₅ is absent.

In one embodiment, X ₆ is serine or asparagine, or X ₆ is absent. In one embodiment, X ₆ is serine or asparagine. In one embodiment, X ₆ is absent.

In one embodiment, X ₁ is selected from the group consisting of lysine, arginine, and conservative amino acid substitutions of any of these; X ₂ is selected from the group consisting of alanine, isoleucine, proline, serine and conservative amino acid substitutions of any one of these; X ₃ is selected from the group consisting of valine, leucine, isoleucine and conservative amino acid substitutions of any one of these; X ₄ is selected from the group consisting of asparagine, glutamic acid, proline and a conservative amino acid substitution of any of the foregoing, or X ₄ is absent; X ₅ is selected from the group consisting of serine, glutamine and conservative amino acid substitutions of any of these, or X ₅ is absent; X ₆ is serine or a conservative amino acid substitution thereof, or X ₆ is absent.

In one embodiment, X ₁ is absent or selected from the group consisting of lysine, arginine, and conservative amino acid substitutions of any one thereof; X ₂ is selected from the group consisting of alanine, isoleucine, proline, serine and conservative amino acid substitutions of any one of these; X ₃ is selected from the group consisting of valine, leucine, isoleucine and conservative amino acid substitutions of any one of these; X ₄ is selected from the group consisting of asparagine, glutamic acid, proline and a conservative amino acid substitution of any of the foregoing, or X ₄ is absent; X ₅ is selected from the group consisting of serine, glutamine and conservative amino acid substitutions of any of these, or X ₅ is absent; X ₆ is serine or a conservative amino acid substitution thereof, or X ₆ is absent.

In one embodiment, X ₁ is lysine or arginine; X ₂ is selected from the group consisting of alanine, isoleucine, proline and serine; X ₃ is selected from the group consisting of valine, leucine and isoleucine; X ₄ is asparagine, proline or glutamic acid, or X ₄ is absent; X ₅ is serine or glutamine or X ₅ is absent; X ₆ is serine or X ₆ is absent.

In one embodiment, X ₁ is absent, or X ₁ is lysine or arginine; X ₂ is selected from the group consisting of alanine, isoleucine, proline and serine; X ₃ is selected from the group consisting of valine, leucine and isoleucine; X ₄ is asparagine, proline or glutamic acid, or X ₄ is absent; X ₅ is serine or glutamine or X ₅ is absent; X ₆ is serine or X ₆ is absent.

In one embodiment, the peptide comprises an amino acid sequence selected from the group consisting of RAL, RALN, RALNS, RALNSS, RSV, RSVE, RSVEG, RSVEGS, RPV, RPVE, RPVES, RPVESS, RII, RIIH, RIIHN and RIIHNN .

In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of RAL, RALN, RALNS, RALNSS, RSV, RSVE, RSVEG, RSVEGS, RPV, RPVE, RPVES, RPVESS, RII, RIIH, RIIHN and RIIHNN .

In one embodiment, the peptide comprises the amino acid sequence ALNSS. In one embodiment, the peptide consists of the amino acid sequence ALNSS.

In one embodiment, the peptide comprises the amino acid sequence KAPLPRS. In one embodiment, the peptide consists of the amino acid sequence KAPLPRS.

In one embodiment, the peptide comprises the amino acid sequence RALNSS.

In one embodiment, the peptide consists of the amino acid sequence RALNSS.

In one embodiment, the peptide comprises the amino acid sequence CRALNSSC.

In one embodiment, the peptide consists of the amino acid sequence CRALNSSC.

In one embodiment, the peptide may compete for binding to LanCL with a peptide consisting of the amino acid sequence CRSVEGSCG.

As described elsewhere herein, the inventors have unexpectedly discovered that peptides that are on the order of three amino acids in length and contain the amino acid sequence of Formula (I) will retain biological activity. In embodiments disclosed herein, the peptide is 3 to 19 amino acid residues in length, preferably 3 to 18 amino acid residues in length, preferably 3 to 17 amino acid residues in length, preferably 3 to 16 amino acid residues in length. a length of 3 to 15 amino acid residues, preferably a length of 3 to 14 amino acid residues, preferably a length of 3 to 13 amino acid residues, preferably a length of 3 to 12 amino acids. The length of the residues, preferably 3 to 11 amino acid residues, preferably 3 to 10 amino acid residues, preferably 3 to 9 amino acid residues, preferably 3 to 8 amino acid residues. length, preferably 3 to 7 amino acid residues in length, preferably 3 to 6 amino acid residues in length, preferably 3 to 5 amino acid residues in length, preferably 3 or 4 amino acid residues in length, or Preferably it is three amino acid residues in length. In one embodiment, the peptide is 20 amino acid residues in length. In one embodiment, the peptide is 19 amino acid residues in length. In one embodiment, the peptide is 18 amino acid residues long. In one embodiment, the peptide is 17 amino acid residues in length. In one embodiment, the peptide is 16 amino acid residues in length. In one embodiment, the peptide is 15 amino acid residues in length. In one embodiment, the peptide is 14 amino acid residues in length. In one embodiment, the peptide is 13 amino acid residues in length. In one embodiment, the peptide is 12 amino acid residues in length. In one embodiment, the peptide is 11 amino acid residues long. In one embodiment, the peptide is 10 amino acid residues long. In one embodiment, the peptide is 9 amino acid residues in length. In one embodiment, the peptide is 8 amino acid residues in length. In one embodiment, the peptide is 7 amino acid residues in length. In one embodiment, the peptide is 6 amino acid residues in length. In one embodiment, the peptide is 5 amino acid residues in length. In one embodiment, the peptide is 4 amino acid residues in length. In one embodiment, the peptide is 3 amino acid residues in length.

Peptides described herein may suitably comprise naturally occurring amino acid residues, either proteinogenic or non-proteinogenic. These amino acids generally have L-stereochemistry. Naturally occurring amino acids are listed in Table 1 below.

Table 1

amino acid three letter abbreviation one letter symbol Structure of the side chain (R) in (1) above alanine Ala A -CH ₃ arginine Arg R -(CH ₂ ) ₃ NHC(=N)NH ₂ Asparagine Asn N -CH ₂ CONH ₂ aspartic acid Asp D -CH ₂ CO ₂ H cysteine Cys C -CH ₂ SH glutamine Gln Q -(CH ₂ ) ₂ CONH ₂ glutamic acid Glu E -(CH ₂ ) ₂ CO ₂ H glycine Gly G -H histidine His H -CH ₂ (4-imidazolyl) isoleucine Ile I -CH(CH ₃ )CH ₂ CH ₃ leucine Leu L -CH ₂ CH(CH ₃ ) ₂ Lysine Lys K -(CH ₂ ) ₄ NH ₂ methionine Met M -(CH ₂ ) ₂ SCH ₃ Phenylalanine Phe F -CH ₂ Ph ornithine Orn O -(CH ₂ ) ₃ NH ₂ proline Pro P For the structure of amino acids, refer to formula (2) above. serine Ser S -CH ₂ OH threonine Thr T -CH(CH ₃ )OH tryptophan Trp W -CH ₂ (3-indolyl) tyrosine Tyr Y -CH ₂ (4-hydroxyphenyl) Valine Val V -CH(CH ₃ ) ₂

As used herein, the term “alkyl” refers to a straight or branched chain saturated hydrocarbon group having 1 to 10 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example C _1-6 alkyl including alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. You can have it. Examples of suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n. -Includes, but is not limited to, hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl and decyl.

As used herein, the term “alkenyl” refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more double bonds between carbon atoms. Where appropriate, alkenyl groups may have a specific number of carbon atoms. For example, C2-C6 as in “C2-C6 alkenyl” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, octenyl, nonenyl and decenyl. Including, but not limited to.

As used herein, the term “alkynyl” refers to a straight-chain or branched hydrocarbon group having one or more triple bonds and having 2 to 10 carbon atoms. Where appropriate, alkynyl groups may have a specific number of carbon atoms. For example, C2-C6 as in "C2-C6 alkynyl" includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

As used herein, the term “cycloalkyl” refers to saturated and unsaturated (but not aromatic) cyclic hydrocarbons. Cycloalkyl rings can contain any number of carbon atoms. For example, a 3 to 8 membered cycloalkyl group contains 3, 4, 5, 6, 7 or 8 carbon atoms. Examples of suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, and cyclooctyl.

As used herein, the term “aryl” refers to any stable monocyclic, bicyclic or tricyclic carbon ring system having up to seven atoms in each ring, where at least one ring is aromatic. Examples of such aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, fluorenyl, phenanthrenyl, biphenyl, and binaphthyl.

In one embodiment, the peptide comprises one or more D-amino acids. In another embodiment, one or more of the amino acids of formula (I) are D-amino acids.

As noted elsewhere herein, the inventors have unexpectedly discovered that the peptides described herein will retain biological activity regardless of whether they exist in a cyclic or linear peptide configuration. Accordingly, in one embodiment, the peptide is a linear peptide. In another embodiment, the peptide is a cyclic peptide. Those skilled in the art will be familiar with suitable methods for forming cyclic peptides, examples of which are described in Choi and Joo ( Biomol Ther (Seoul). 2020; 28(1): 18-24), referenced herein. It is included as

In one embodiment, the peptide is cyclized by a disulfide bond between two cysteine residues. In one embodiment, the disulfide bond is formed between two cysteine residues, wherein the two cysteine residues are immediately adjacent to the C-terminal (X ₆ ) and N-terminal (X ₁ ) residues of Formula (I) ; That is, the peptide will contain the amino acid sequence Cysteine- _{X 1} -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -Cysteine. Alternatively, the disulfide bond is formed between two cysteine residues, where one or both of the cysteine residues are distal to the C-terminal (X ₆ ) and N-terminal (X ₁ ) residues of Formula (I). Located. For example, the peptide has the amino acid sequence Cysteine- _{X 1} -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -Cysteine or Cysteine-X ₁ -X 2 -X ₃ -X _{4 -X 5 -X 6} -Y-cysteine or cysteine-YX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -Y-cysteine, where Y is one or more amino acid residues. In another example, the peptide has _the amino acid sequence Cysteine- _{YX 2} -X _{3 -X 4 -X 5 -X 6} -Cysteine or Cysteine- Cysteine-YX ₂ -X ₃ -X ₄ -X ₅ -X ₆ -Y-cysteine, where Y is one or more amino acid residues. In one embodiment, the cyclic peptide is formed by a disulfide bond between two cysteine residues.

As described elsewhere herein, the inventors unexpectedly discovered that certain cyclic peptides comprising the amino acid sequence of formula (I) have greater biological activity when compared to their linear counterparts. For example, we found that the cyclic peptide CQEQLERALNSSC, cyclized by a disulfide bond between two cysteine residues, had greater binding affinity for LanCL and did not cyclize in an animal model of influenza A respiratory tract infection. It has been shown to be more effective in vivo when compared to its uncomplicated counterpart, QEQLERALNSS. Accordingly, in one embodiment, the cyclic peptide comprises the amino acid sequence CQEQLERALNSSC. In one embodiment, the cyclic peptide consists of the amino acid sequence CQEQLERALNSSC.

The peptides described herein can be prepared by suitable methods well known to those skilled in the art, examples of which include solution or solid phase synthesis using Fmoc or Boc protected amino acid residues and standard microbial culture techniques, genetically engineered microorganisms and recombinant DNA techniques. Recombinant techniques known in the art to be used are included (Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed.), 2001, CSHL Press).

In one embodiment, the peptides described herein are formed into pharmaceutically acceptable salts. It should be understood that non-pharmaceutically acceptable salts are also contemplated, as they may be useful as intermediates in the preparation of pharmaceutically acceptable salts or during storage or transportation. Suitable pharmaceutically acceptable salts will be familiar to those skilled in the art and examples include salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid and hydrobromic acid, or acetic acid, propionic acid, Butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, maleic acid, citric acid, lactic acid, mucinic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid sulfa. Included are salts of pharmaceutically acceptable inorganic acids such as nitric acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valeric acid. Examples of suitable base salts include salts formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. Basic nitrogen-containing groups include lower alkyl halides (such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides); dialkyl sulfates such as dimethyl and diethyl sulfate; It can be quaternized with reagents such as:

Also disclosed herein are prodrugs comprising the peptides described herein or pharmaceutically acceptable salts thereof. As used herein, “prodrug” generally refers to a compound that can be metabolized in vivo to provide or release the active peptide described herein or a pharmaceutically acceptable salt thereof. In one embodiment, the prodrug itself also shares the same or substantially the same therapeutic activity as the peptide described herein or a pharmaceutically acceptable salt thereof as described elsewhere herein.

In some embodiments, the peptides described herein or pharmaceutically acceptable salts thereof may further comprise a C-terminal capping group. As used herein, the term “C-terminal capping group” refers to a group that blocks the reactivity of the C-terminal carboxylic acid. Suitable C-terminal capping groups form amide groups or esters with the C-terminal carboxylic acid, for example, C-terminal capping groups form -C(O)NHR ^a or -C(O)OR ^b , where C (O) is derived from a C-terminal carboxylic acid group, R ^a is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl, and R ^b is alkyl, alkenyl, alkynyl, cycloalkyl or aryl. In certain embodiments, the C-terminal capping group is -NH ₂ , forming -C(O)NH ₂ . In some embodiments, the peptides described herein or pharmaceutically acceptable salts thereof comprise a C-terminal polyethylene glycol (PEG). In one embodiment, PEG has a molecular weight ranging from 220 to 5500 Da, preferably from 220 to 2500 Da, more preferably from 570 to 1100 Da.

In some embodiments, the peptides described herein or pharmaceutically acceptable salts thereof may further comprise an N-terminal capping group. As used herein, the term “N-terminal capping group” refers to a group that blocks the reactivity of the N-terminal amino group. A suitable N-terminal capping group is an acyl group which together with the N-terminal amino group forms an amide group, for example an N-terminal capping group forms -NHC(O)R ^a where NH is derived from the N-terminal amino group and R ^a is alkyl, alkenyl, alkynyl, cycloalkyl, or aryl. In certain embodiments, the N-terminal capping group is -C(O)CH ₃ (acyl), forming -NHC(O)CH ₃ .

In some embodiments, a peptide described herein or a pharmaceutically acceptable salt thereof may comprise a C-terminal capping group and an N-terminal capping group as described herein. It should be understood that the peptides disclosed herein do not include the full-length amino acid sequence of human growth hormone or its non-human isoforms.

Treatment and prevention methods

As described elsewhere herein, the inventors have surprisingly discovered that the peptides described herein have advantageous properties that make them useful for therapeutic applications, including treating conditions associated with cellular aging, damage, and stress. Illustrative examples of such conditions include aging, pain, inflammatory conditions/inflammation, and microbial infections. The activities imparted to the peptides described herein also make them useful as anti-aging compounds. Accordingly, the peptides described herein can suitably be used to treat, alleviate, or otherwise eliminate the severity of such conditions in an individual in need thereof, including one or more symptoms. Accordingly, the present disclosure extends to a method of treating a condition in an individual, comprising administering a therapeutically effective amount of a peptide described herein to an individual in need thereof. Also provided is the use of the peptides described herein in the manufacture of a medicament for the treatment of a condition in an individual in need thereof. Also provided are the peptides described herein for use in the treatment of a condition in an individual in need thereof.

In one embodiment, the condition includes pain, inflammatory airway disease, microbial infection, respiratory tract infection, migraine, sarcopenia, impaired glucose tolerance, diabetes, obesity, metabolic disease and obesity-related disease, osteoarthritis, muscle disorder, wasting disorder, aging, cachexia. , including anorexia, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, amyotrophic lateral sclerosis (ALS), motor neurone disease, neuromuscular junction disease, ophthalmic condition, central nervous system condition, and neurodegenerative condition (e.g., Parkinson's disease, Alzheimer's disease). , inflammatory myopathies, burns, wounds, injuries or trauma, conditions associated with elevated LDL cholesterol, conditions associated with damaged chondrocytes, proteoglycans or collagen production or quality, conditions associated with impaired cartilaginous tissue formation or quality, damaged muscles, ligaments or tendons. selected from the group consisting of conditions associated with mass, form or function, inflammation, trauma or genetic abnormalities affecting muscle or connective tissue, and bone disorders.

The terms “treat,” “treatment,” and the like are used interchangeably herein to mean alleviating, reducing, alleviating, ameliorating or inhibiting the severity of a disease or condition, including one or more symptoms. The terms “treating,” “treatment,” and the like are also used interchangeably herein to include preventing a disease or condition, including one or more symptoms.

The terms “treat,” “treatment,” and the like also include preventing, alleviating, reducing, alleviating, ameliorating or inhibiting the severity of a disease, condition and/or one or more symptoms thereof for at least a period of time. The terms “treating,” “treatment,” and the like mean that the disease, condition, or one or more symptoms thereof is prevented, alleviated, reduced, alleviated, ameliorated, or suppressed permanently, thereby temporarily preventing the severity of the disease, condition, or one or more symptoms thereof; It should be understood that it does not mean that it extends to alleviating, reducing, alleviating, alleviating or suppressing.

As used herein, the term “subject” refers to a mammalian individual for whom treatment of a disease, condition, or one or more symptoms thereof is desired. Illustrative examples of suitable subjects include primates, especially humans, companion animals such as cats and dogs, working animals such as horses, donkeys, etc., domestic animals such as sheep, cows, goats, pigs, etc., rabbits, mice, rats, guinea pigs, hamsters, etc. This includes laboratory test animals, those in zoos, wildlife parks, and captive wild animals such as deer, dingoes, etc. In one embodiment, the individual is a human.

It should be understood that reference to an individual herein does not mean that the individual has a disease, condition, or one or more symptoms, but also includes individuals at risk of developing the disease, condition, or one or more symptoms.

In one embodiment, the methods disclosed herein include administering a peptide disclosed herein, or a pharmaceutically acceptable salt thereof, to a human subject.

It should be understood that the peptides described herein, or pharmaceutically acceptable salts thereof, are advantageously administered in therapeutically effective amounts. The phrase “therapeutically effective amount” generally refers to the amount necessary to achieve the desired response. Those skilled in the art will appreciate that the therapeutically effective amount of a peptide will depend on a number of factors, including, but not limited to, the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the severity of the disease, condition or condition being treated, and the peptide or its pharmacology described herein. Formulations of compositions containing commercially acceptable salts, routes of administration, and combinations of any of the foregoing.

Therapeutically effective amounts will generally fall within relatively broad ranges that can be determined through routine trial by those skilled in the art. Illustrative examples of therapeutically effective amounts of the peptides described herein and pharmaceutically acceptable salts thereof suitable for administration to a human subject include from about 0.001 mg per kg body weight to about 1 g per kg body weight, preferably about 0.001 mg per kg body weight. to about 50 g per kg of body weight, more preferably from about 0.01 mg per kg to about 1.0 mg per kg of body weight. In one embodiment disclosed herein, the therapeutically effective amount of a peptide described herein and/or a pharmaceutically acceptable salt thereof is from about 0.001 mg per kg body weight to about 1 g per kg body weight per dose (e.g., 0.001 mg per kg body weight). mg/kg, 0.005mg/kg, 0.01mg/kg, 0.05mg/kg, 0.1mg/kg, 0.15mg/kg, 0.2mg/kg, 0.25mg/kg, 0.3mg/kg, 0.35mg/kg, 0.4 mg/kg, 0.45mg/kg, 0.5mg/kg, 0.5mg/kg, 0.55mg/kg, 0.6mg/kg, 0.65mg/kg, 0.7mg/kg, 0.75mg/kg, 0.8mg/kg, 0.85 mg/kg, 0.9mg/kg, 0.95mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 2.5mg/kg, 3mg/kg, 3.5mg/kg, 4mg/kg, 4.5mg/kg, 5mg/kg, 5.5mg/kg, 6mg/kg, 6.5mg/kg, 7mg/kg, 7.5mg/kg, 8mg/kg, 8.5mg/kg, 9mg/kg, 9.5mg/kg, 10mg/kg, 10.5 mg/kg, 11mg/kg, 11.5mg/kg, 12mg/kg, 12.5mg/kg, 13mg/kg, 13.5mg/kg, 14mg/kg, 14.5mg/kg, 15mg/kg, 15.5mg/kg, 16mg /kg, 16.5mg/kg, 17mg/kg, 17.5mg/kg, 18mg/kg, 18.5mg/kg, 19mg/kg, 19.5mg/kg, 20mg/kg, 20.5mg/kg, 21mg/kg, 21.5mg / kg, 22mg/kg, 22.5mg/kg, 23mg/kg, 23.5mg/kg, 24mg/kg, 24.5mg/kg, 25mg/kg, 25.5mg/kg, 26mg/kg, 26.5mg/kg, 27mg/ kg, 27.5mg/kg, 28mg/kg, 28.5mg/kg, 29mg/kg, 29.5mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg /kg, 65mg/kg, 70mg/kg, 75mg/kg, 80mg/kg, 85mg/kg, 90mg/kg, 95mg/kg, 100mg/kg, 105mg/kg, 110mg/kg, etc.). In one embodiment, a therapeutically effective amount of a peptide described herein or a pharmaceutically acceptable salt thereof is from about 0.001 mg to about 50 mg per kg of body weight. In one embodiment, the therapeutically effective amount of the peptides described herein and pharmaceutically acceptable salts thereof is from about 0.01 mg to about 100 mg per kg of body weight. In one embodiment, the therapeutically effective amount of a peptide described herein or a pharmaceutically acceptable salt thereof is from about 0.1 mg to about 10 mg per kg of body weight, preferably from about 0.1 mg to about 5 mg per kg of body weight, more preferably It ranges from about 0.1 mg to about 1.0 mg per kg of body weight. Dosage regimens may be adjusted to provide optimal therapeutic response. For example, several divided doses may be administered daily, weekly, monthly, or at other appropriate time intervals, or the dose may be reduced proportionally as the urgency of the situation dictates.

ache

As described elsewhere herein, the inventors have discovered that the peptides described herein have advantageous analgesic properties, including alleviating neuropathic pain. Accordingly, in one embodiment, the condition is pain. In one embodiment, the condition is neuropathic pain.

Without being bound by theory or specific application, neuropathic pain is generally characterized as pain resulting from damage or disease to the nerve tissue or neurons themselves, or due to dysfunction within the nerve tissue. Pain may be peripheral, central, or a combination thereof; in other words, the term “neuropathic pain” generally refers to any pain syndrome that begins or results from a primary lesion or dysfunction of the peripheral or central nervous system. Neuropathic pain is also distinct in that it generally does not respond effectively to treatment with common analgesics such as opioids. In contrast, nociceptive pain is characterized as pain resulting from stimulation of nociceptors by noxious or potentially noxious stimuli that can cause damage or scarring of tissues. Nociceptive pain usually responds to common analgesics such as opioids.

The term “analgesia” is used herein to describe conditions in which there is reduced or absent sensitivity to noxious stimuli, including the absence of pain sensation, as well as conditions in which pain perception is reduced. This condition, with reduced or absent pain perception, is typically induced by the administration of a pain modifier or agents and occurs without loss of consciousness, as is generally understood in the art. Suitable methods for determining whether a compound may provide analgesic effects will be familiar to those skilled in the art, examples of which include chronic constriction injuries, spinal nerve ligation, and partial sciatic nerve ligation (Bennett et al. (2003); Curr. Protoc. Neurosci ., Chapter 9, Unit 9.14) and the use of animal models of nociceptive pain, such as formalin-, carrageenan-, or complete Freund's adjuvant (CFA)-induced inflammatory pain. Other suitable models of pain are discussed in Gregory et al . (2013, J. Pain .; 14(11); ": An overview of animal models of pain: disease models and outcome measures ".

As those skilled in the art will appreciate, the causes of neuropathy and neuropathic pain can vary. Accordingly, it should be understood that treatment or prevention of neuropathic pain, regardless of cause, is contemplated herein. In some embodiments, neuropathic pain is the result of a disease or condition affecting the nerves (primary neuropathy) and/or neuropathy caused by a systemic disease (secondary neuropathy), illustrative examples of which are diabetic neuropathy; Neuropathy associated with herpes zoster (herpes zoster); fibromyalgia; Multiple sclerosis, stroke, spinal cord injury; Chronic post-operative pain, phantom limb pain, Parkinson's disease; uremia-related neuropathy; amyloidosis neuropathy; HIV sensory neuropathy; Hereditary motor and sensory neuropathy (HMSN); Hereditary sensory neuropathy (HSN); Hereditary sensory and autonomic neuropathy; Hereditary neuropathy with ulcerative amputation; nitrofurantoin neuropathy; neoplastic neuropathy; These include neuropathy due to nutritional deficiency, neuropathy due to renal failure, and complex regional pain syndrome. Other examples of conditions that can cause neuropathic pain include repetitive activities such as typing or assembly line work, and several antiretroviral drugs known to cause peripheral neuropathy, such as zalcitabine (ddC) and didanosine (ddI). These include known drugs, antibiotics (metronidazole, an antibiotic used for Crohn's disease, isoniazid, used for tuberculosis), gold compounds (used for rheumatoid arthritis), some chemotherapy drugs (such as vincristine, etc.), and many others. Chemical compounds, including alcohol, lead, arsenic, mercury, and organophosphate pesticides, are also known to cause peripheral neuropathy. Some peripheral neuropathies are associated with infectious processes (e.g. Guillain-Barre syndrome). Other illustrative examples of neuropathic pain include thermal or mechanical hyperalgesia, thermal or mechanical allodynia, diabetic pain, neuropathic pain affecting the oral cavity (e.g., trigeminal neuropathic pain, atypical dental pain (phantom odontodynia)) , burning mouth syndrome), fibromyalgia, and entrapment pain. .

In one embodiment disclosed herein, the neuropathic pain includes diabetic neuropathy; Neuropathy associated with shingles; fibromyalgia; Multiple sclerosis, stroke, spinal cord injury; Chronic post-operative pain, phantom limb pain, Parkinson's disease; uremia-related neuropathy; amyloidosis neuropathy; HIV sensory neuropathy; Hereditary motor and sensory neuropathy (HMSN); Hereditary sensory neuropathy (HSN); Hereditary sensory and autonomic neuropathy; Hereditary neuropathy with ulcerative amputation; nitrofurantoin neuropathy; neoplastic neuropathy; Neuropathy due to nutritional deficiency, neuropathy due to renal failure, trigeminal neuropathic pain, atypical dental pain (phantom toothache), burning mouth syndrome, complex regional pain syndrome, repetitive strain injury, drug-induced peripheral neuropathy, infection, allodynia, It is selected from the group consisting of peripheral neuropathies associated with hyperesthesia, hyperalgesia, burning pain and shooting pain.

In some embodiments, neuropathic pain may be accompanied by numbness, weakness, and loss of reflexes. The pain can be severe and disabling. “Hyperalgesia” generally means an increased response to painful stimuli. A hyperalgesic condition is a condition associated with pain caused by a normally non-painful stimulus. The term “hyperesthesia” refers to excessive physical sensitivity, especially of the skin. As used herein, the term “allodynia” refers to pain caused by non-noxious stimuli; In other words, it is pain caused by a stimulus that does not normally cause pain. Illustrative examples of allodynia include thermal allodynia (pain caused by cold or hot stimuli), tactile allodynia (pain caused by light pressure or touch), and mechanical allodynia (pain caused by strong pressure or pinpricks).

Neuropathic pain can be acute or chronic, and in this context it should be understood that the time course of neuropathy may vary depending on the underlying cause. For example, in the case of trauma, the onset of neuropathic pain or symptoms of neuropathic pain may be acute or sudden. However, the most severe symptoms develop over time and can last for years. A chronic course of several weeks to months usually indicates toxic or metabolic neuropathy. Chronic, slowly progressive neuropathies, such as those that occur in painful diabetic neuropathy, most inherited neuropathies, or a condition called chronic inflammatory demyelinating polyradiculopathy (CIDP), can progress over several years. Neuropathic conditions with relapsing and remitting symptoms include Guillain-Barré syndrome.

In some embodiments, neuropathic pain results from a condition characterized by nervous hypersensitivity, such as fibromyalgia or irritable bowel syndrome.

In other embodiments, neuropathic pain results from disorders associated with abnormal nerve regeneration resulting in nerve hypersensitivity. These disorders include breast pain, interstitial cystitis, vulvodynia, and cancer chemotherapy-induced neuropathy.

In some embodiments, neuropathic pain is associated with surgery, pre-operative pain and postoperative pain, particularly postoperative neuropathic pain.

microbial infection

Microbial infections caused by pathogens such as bacteria, viruses and fungi remain a major global health problem with significant socioeconomic costs. Treatment of bacterial infections relies primarily on antibiotics, while the standard approach for viral infections remains supportive care and symptom relief. While these treatments have shown some efficacy, newly emerging or re-emerging pathogens continue to plague human and non-human populations, at least in part due to their resistance to existing pharmacological interventions and/or new emerging pathogens with enhanced infectivity. It is caused by mutations that create variants. The lack of timely available antiviral drugs, including vaccines, has also made it difficult to contain the virus outbreak globally.

More than 200 serological strains of viruses that cause infections, including respiratory tract infections, are known, the most common of which is rhinovirus (30-50%). Others include coronaviruses (10-15%), influenza (5-15%), human parainfluenza viruses, human respiratory syncytial viruses, adenoviruses, enteroviruses, and metapneumoviruses. More than 30 coronaviruses have been identified, but only three or four are known to cause respiratory tract infections in humans. Moreover, coronaviruses are generally difficult to culture in vitro, making it difficult to study their function and develop appropriate treatments. Coronaviruses are enveloped positive-strand RNA viruses that originate in the endoplasmic reticulum-Golgi intermediate compartment or cis-Golgi network. Coronaviruses infect humans and animals. Human coronaviruses 229E, OC43, and the recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; see Zhu N et al .. N Engl J Med . 2020) are known to be major causes of respiratory tract infections; It can cause pneumonia, especially in the elderly, newborns, and immunocompromised people. Illustrative examples of coronaviruses causing respiratory tract infections are described in U.S. Patent Publication No. 20190389816, the contents of which are incorporated herein by reference in their entirety.

Another widespread viral infection is caused by human rhinovirus (HRV), a member of the Enterovirus genus of the Picornaviridae family. HRV can cause cold symptoms by infecting the upper and lower respiratory tract, including the nasal mucosa, paranasal sinuses, and middle ear. Infection is usually self-limiting and limited to the upper respiratory tract.

Some viral infections cause no symptoms in some people but are contagious to others. In these cases, spread of the virus can spread widely because infected people do not appear sick. Transmission is particularly harmful in schools, hospitals, nursing homes and other areas where vulnerable populations live in close proximity.

Currently, few antiviral drugs are approved for the treatment or prevention of respiratory viral infections, including the flu or colds. These include oseltamivir phosphate (brand name Tamiflu®), zanamivir (brand name Relenza®), peramivir (brand name Rapivab®), and baloxavir marboxil (brand name Xofluza®). Treatment of respiratory tract infections usually involves reducing symptoms (sneezing, stuffy nose, runny nose, eye irritation, sore throat, cough, headache, fever, chills), such as over-the-counter oral antihistamines, aspirin, cough suppressants, and decongestants. Management is generally based. Symptomatic treatment usually involves taking antihistamines and/or vasoconstrictor decongestants, many of which have undesirable side effects such as drowsiness.

Without wishing to be bound by theory or particular application, the present inventors have surprisingly discovered that the peptides described herein can be used to treat microbial infections, including alleviating at least some of the symptoms of infections such as respiratory tract infections.

Respiratory tract infection (RTI) is generally defined as any infectious disease of the upper or lower respiratory tract. Upper respiratory tract infections (URTIs) include colds, laryngitis, pharyngitis/tonsillitis, acute rhinitis, acute rhinosinusitis, and acute otitis media. Lower respiratory tract infections (LRTIs) include acute bronchitis, bronchiolitis, pneumonia, and tracheitis. Antibiotics are commonly prescribed for RTIs in adults and children receiving primary care. RTIs account for 60% of all antibiotics prescribed in general practice and result in significant costs to the healthcare system ( NICE Clinical Guidelines, No. 69 ; Center for Clinical Practice at NICE (UK), London: National Institute for Health and Clinical Excellence (UK); 2008) .

Pathogens that cause upper and/or lower respiratory tract infections in humans and non-human subjects will be known to those skilled in the art and include bacteria and viruses, examples of which are described in Charlton et al. ( Clinical Microbiology Reviews ; 2018, 32 (1): e00042 -18), Popescu et al . ( Microorganisms . 2019; 7(11): 521) and Kikkert, M. ( J Innate Immun . 2020; 12(1): 4-20), the contents of which are published in their entirety. The content is incorporated herein by reference. In one embodiment, the respiratory tract infection is a viral infection.

Viruses that cause infections of the respiratory tract (upper and/or lower respiratory tract) in humans and non-human subjects will be known to those skilled in the art and include, for example, picornaviruses, coronaviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, These include adenoviruses, enteroviruses, and metapneumoviruses. Accordingly, in one embodiment disclosed herein, the virus is selected from the group consisting of picornavirus, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, enterovirus, and metapneumovirus. In one embodiment, the virus is an influenza virus. In another embodiment, the virus is a coronavirus. Illustrative examples of coronaviruses that cause respiratory tract infections will be familiar to those skilled in the art, including SARS-CoV-2, previously described in Zhu N et al., (N Engl J Med. 2020) and US Patent Publication No. 20190389816. and is incorporated herein by reference in its entirety. In one embodiment, the virus is SARS-CoV-2.

The peptides described herein may be particularly useful in treating respiratory tract infections in individuals with underlying medical conditions that may exacerbate respiratory tract infections. These underlying conditions will be known to those skilled in the art and examples include chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema and lung cancer. In one embodiment, the individual has an additional respiratory condition selected from the group consisting of chronic obstructive pulmonary disease, asthma, cystic fibrosis, and lung cancer. In another embodiment, the individual is immunocompromised as a result of treatment (e.g., by chemotherapy, radiotherapy) or other methods (e.g., HIV infection).

Viral replication of viruses in humans generally begins 2 to 6 hours after initial contact. In some cases, patients are contagious for several days before symptoms appear. Symptoms usually begin about 2 to 5 days after initial infection. Respiratory tract infections, such as the common cold, are most contagious during the first two to three days after symptoms appear. There is currently no known treatment that shortens the duration of a cold. Although symptoms usually go away on their own in about 7 to 10 days, some symptoms may last up to 3 weeks. The virus may still be contagious until symptoms have completely resolved.

As noted elsewhere herein, the inventors have also discovered that the peptides described herein are surprisingly effective in limiting viral replication in vivo and reducing hyperinflammation and severe disease during IAV infection.

Inflammatory airway disease

In embodiments disclosed herein, the condition is an inflammatory airway disease. Inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD), asthma, chronic bronchitis, emphysema, cystic fibrosis, lung cancer and bronchopulmonary dysplasia are among the most prevalent diseases in the world. In particular, the prevalence of asthma has increased over the past two decades and now affects up to 10% of the population in most developed countries. COPD is the sixth most common cause of death in the world and is known to affect approximately 4-6% of people over 45 years of age. It is indisputable that inflammatory airway diseases pose a significant financial burden to society when both direct and indirect costs are taken into account.

Asthma and COPD are identified by the presence of characteristic symptoms and functional abnormalities, and airway obstruction is an essential component of both diseases. While the airway obstruction in asthma is generally reversible, COPD is typically characterized by abnormal expiratory flow that does not change appreciably over several months of observation. Both airway diseases are associated with lung inflammation triggered by different initiating factors, examples of which include environmental allergens and carcinogens, occupational sensitizers, tobacco smoke, asbestos, and silica. However, it should be noted that some non-smoking asthma patients may develop irreversible airway obstruction similar to COPD.

Chronic obstructive pulmonary disease is a growing health care problem that is expected to worsen as the population ages and the use of tobacco products increases globally. Quitting smoking is the only effective prevention method. Employers are in a unique position to help their employees quit smoking. During the long asymptomatic phase, lung function nevertheless continues to decline. Therefore, many patients only seek medical attention in advanced stages or when experiencing an acute exacerbation. To maintain patients' quality of life and reduce healthcare costs associated with this chronic disease, clinicians must accurately diagnose the condition and appropriately manage patients during the long term of the disease.

As noted by Devine, FJ (2008; Am Health Drug Benefits; 1(7):34-42), COPD is a poorly reversible lung disease and is one of the leading causes of morbidity and mortality worldwide. In contrast to other major chronic disease trends in the United States, COPD prevalence and mortality have continued to increase, with mortality doubling between 1970 and 2002, and the mortality rate in women now exceeding that in men. Considering that most COPD cases are caused by smoking, this is a fundamentally preventable disease. Most COPD patients are middle-aged or elderly. Effective treatments for COPD remain largely elusive. The only strategy known to reduce the incidence of the disease is to stop smoking.

Asthma is a heterogeneous, multifactorial disease with variable and mostly reversible respiratory pathway obstruction based on a chronic bronchial inflammatory response (Horak et al., 2016; Wien Klin Wochenschr. 128(15):541-554). Symptoms of asthma (coughing, phlegm, whistling, wheezing, chest tightness, or shortness of breath) vary and are usually associated with restricted expiratory flow. Due to the heterogeneity of asthma, a variety of phenotypes can be attributed to asthma, including: allergic asthma, non-allergic asthma, pediatric asthma/recurrent obstructive bronchitis, late-onset asthma, asthma with fixed airflow obstruction, obesity- Associated asthma, occupational asthma, geriatric asthma and severe asthma.

Asthma treatment (pharmacological and non-pharmacological interventions) is primarily based on symptom control (cycle of assessment, intervention and review) and is usually associated with reduction of asthma exacerbations. From a pharmacological perspective, the gold standard for asthma treatment is generally low-dose inhaled corticosteroids, often combined with on-demand short-acting beta-2 agonists (SABAs). Other treatments include combinations of leukotriene receptor antagonists (LTRAs), low-dose inhaled corticosteroids, and long-acting beta-2 agonists (LABAs). However, existing treatments have the potential to cause side effects, especially when used long-term. Common side effects of preventive medications (such as inhaled corticosteroids) include hoarseness, sores in the mouth and throat, and fungal infections of the throat.

The present inventors have surprisingly discovered that the peptides described herein can alleviate at least some of the inflammatory mediators of inflammatory airway diseases.

Inflammatory airway diseases will be familiar to those skilled in the art and examples include chronic obstructive pulmonary disease (COPD), asthma, chronic bronchitis, emphysema, cystic fibrosis, lung cancer, and bronchopulmonary dysplasia. In one embodiment, the inflammatory airway disease is COPD. In one embodiment, the inflammatory airway disease is asthma. In one embodiment, the inflammatory airway disease is chronic bronchitis. In one embodiment, the inflammatory airway disease is emphysema. In one embodiment, the inflammatory airway disease is cystic fibrosis. In one embodiment, inflammatory airway disease is associated with lung cancer. In one embodiment, the inflammatory airway disease is bronchopulmonary dysplasia.

The methods described herein may be particularly useful for treating inflammatory airway disease in individuals susceptible to conditions that may exacerbate inflammatory airway disease. Such underlying conditions will be known to those skilled in the art and examples include, for example, respiratory infections caused by viruses, bacteria or other pathogens. In another embodiment, the individual is immunocompromised as a result of treatment (e.g., by chemotherapy, radiotherapy) or other methods (e.g., HIV infection).

Route of administration

Peptides and pharmaceutically acceptable salts thereof as described herein can be administered to an individual by any suitable route allowing delivery of the peptide or pharmaceutically acceptable salt thereof to the individual in a therapeutically effective amount as described herein. there is. Suitable routes of administration will be known to those skilled in the art, and illustrative examples include enteral routes of administration (e.g., oral and rectal), parenteral routes of administration, typically by injection or microinjection (e.g., intramuscular, subcutaneous, intravenous, epidural, intraarticular, intraperitoneal, intracisternal, or intrathecal) and topical (transdermal or transmucosal) routes of administration (e.g., buccal, sublingual, vaginal, intranasal, or by inhalation, insufflation, suppository, or spray). pursuant to). In one embodiment, the route of administration is by inhalation or insufflation. The peptides and pharmaceutically acceptable salts thereof as described herein may also be suitably administered to a subject as controlled release dosage forms to provide controlled release of the active agent(s) over an extended period of time. The term “controlled release” typically refers to a release over a period of time (e.g., from about 8 hours up to about 12 hours, up to about 14 hours, up to about 16 hours, up to about 18 hours, up to about 20 hours, up to a day, up to a week, means the release of the active substance(s) to provide a constant or substantially constant concentration of the active substance to the subject over a period of time (up to one month or more than one month). Controlled release of the active agent(s) may begin within minutes after administration or, as needed, within minutes after administration or after expiration of the lag time after administration. Suitable controlled release dosage forms will be known to those skilled in the art, examples of which are described in Anal, A.K. (2010; Controlled Release Dosage Forms. Encyclopedia of Pharmaceutical Sciences. 11:1-46).

Without being bound by theory or particular mode of application, it may be desirable to select the route of administration based on the severity of the disease, condition, or one or more symptoms thereof, as described herein. In one embodiment disclosed herein, a peptide described herein, or a pharmaceutically acceptable salt thereof, is administered enterically to a subject. In one embodiment disclosed herein, the peptide described herein or a pharmaceutically acceptable salt thereof is administered orally to a subject. In one embodiment disclosed herein, a peptide described herein or a pharmaceutically acceptable salt thereof is administered parenterally to an individual. In another embodiment disclosed herein, a peptide described herein, or a pharmaceutically acceptable salt thereof, is administered topically to an individual. In another embodiment disclosed herein, a peptide described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject by inhalation. In another embodiment disclosed herein, a peptide described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject by insufflation.

As described elsewhere herein, “topical” administration typically refers to creams, lotions, foams, gels, ointments, nasal drops, eye drops, ear drops, transdermal patches, transdermal films, suitable for use on body surfaces such as the skin or mucous membranes. This means applying the active agent in the form of a sublingual film (e.g., sublingual film). Topical administration also includes administration through the airway mucosa by inhalation or insufflation. In one embodiment disclosed herein, the topical administration is selected from the group consisting of transdermal and transmucosal administration. In one embodiment, the peptide as described herein, or a pharmaceutically acceptable salt thereof, is administered transdermally to the individual. In one embodiment, a peptide as described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject by inhalation, insufflation, or nebulization.

In one embodiment, the method comprises administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a human by inhalation or insufflation. In another embodiment, the method comprises administering a peptide or a pharmaceutically acceptable salt thereof as described herein to a non-human individual by inhalation or insufflation. In another embodiment, the method comprises administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a non-human individual selected from the group consisting of feline, canine, and equine.

In one embodiment, the method comprises orally administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a human. In another embodiment, the method comprises orally administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a non-human subject. In another embodiment, the method comprises orally administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a non-human individual selected from the group consisting of feline, canine, and equine.

Illustrative examples of topical administration are described elsewhere herein. In one embodiment, the topical administration is transdermal administration.

In one embodiment disclosed herein, the peptides described herein, or pharmaceutically acceptable salts thereof, are administered to a subject in a controlled release dosage form, examples of which are described elsewhere herein. In one embodiment, the method comprises administering to a human a peptide as described herein, or a pharmaceutically acceptable salt thereof, in a controlled release dosage form. In another embodiment, the method comprises administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, to a non-human subject in a controlled release dosage form. In another embodiment, the method comprises administering a peptide as described herein, or a pharmaceutically acceptable salt thereof, in a controlled release dosage form to a non-human individual selected from the group consisting of feline, canine, and equine.

As noted elsewhere herein, multiple (i.e., multiple) divided doses may be administered at daily, weekly, monthly, or other suitable time intervals, or the doses may be reduced proportionally depending on the urgency of the situation. When multiple administration procedures are necessary or otherwise desired, it may be advantageous to administer the peptide via more than one route as disclosed herein. For example, the primary dose may be administered parenterally (e.g., via intramuscular, intravenous, subcutaneous, epidural, intra-articular, intraperitoneal, intracisternal, or intrathecal routes of administration) to induce a rapid or acute therapeutic effect in the subject. ), followed by subsequent (e.g., second, third, fourth, fifth, etc.) doses enterally (e.g., orally or rectally), inhaled or inhaled, and/or topically (e.g., transdermal or transmucosal administration). It may be desirable to provide continued availability of the active agent over a long period of time after the acute treatment phase. Alternatively, a dose may be administered enterally (e.g., orally or rectally), followed by subsequent (e.g., second, third, fourth, fifth, etc.) doses parenterally (e.g., intramuscularly, intravenously, subcutaneously, etc.). It may be desirable to administer by inhalation or insufflation (e.g., epidural, intra-articular, intraperitoneal, intracisternal or intrathecal routes of administration) and/or topically (e.g., via transdermal or transmucosal routes of administration). . Alternatively, a dose may be administered topically (e.g., via transdermal or transmucosal routes of administration) followed by subsequent (e.g., second, third, fourth, fifth, etc.) doses parenterally (e.g., via transdermal or transmucosal routes of administration). It may be desirable to administer by inhalation or insufflation (e.g., via intramuscular, intravenous, subcutaneous, epidural, intra-articular, intraperitoneal, intracisternal or intrathecal routes of administration) and/or enterally (e.g. orally or rectally). You can.

Additionally, it should be understood that when multiple routes of administration are desired, any combination of two or more routes of administration may be used according to the methods disclosed herein. Illustrative examples of suitable combinations include (in order of administration): (a) parenteral-enteral; (b) parenteral-topical; (c) parenteral-enteral-topical; (d) parenteral-topical-enteral; (e) enteral-parenteral; (f) enteral-topical; (g) enteral-topical-parenteral; (h) enteral-parenteral-topical; (i) Topical-parenteral; (j) local-enteral; (k) topical-parenteral-enteral; (l) Topical-enteral-parenteral; (m) parenteral-enteral-topical-parenteral; (n) parenteral-enteral-topical-enteral; Including, but not limited to, etc.

pharmaceutical composition

The peptides or pharmaceutically acceptable salts thereof as described herein can be formulated for administration to a subject as pure chemicals. However, in certain embodiments, it may be desirable to formulate a peptide as described herein, or a pharmaceutically acceptable salt thereof, into pharmaceutical compositions, including veterinary compositions. Accordingly, in another aspect disclosed herein, a peptide described herein is provided for use in treating a condition in an individual in need thereof.

As noted elsewhere herein, the peptides described herein and pharmaceutically acceptable salts thereof may be administered sequentially or in combination (e.g., as a mixture) with one or more other active agents suitable for the underlying condition being treated. there is. For example, the compositions disclosed herein may be formulated for administration together with inhaled corticosteroids commonly used to treat asthma, either sequentially or in combination (e.g., as a mixture). Other suitable combinations or adjuvant treatments will be familiar to those skilled in the art, and the choice will depend on the underlying condition or symptoms thereof.

In one embodiment, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent as described elsewhere herein.

Peptides and pharmaceutically acceptable salts thereof as described herein can be administered as oral solids (e.g., tablets or filled capsules) or liquids (e.g., solutions, suspensions, emulsions, elixirs, or capsules containing them). ) in the form of ointments, suppositories or enemas for rectal administration, in the form of sterile injectable solutions for parenteral use (e.g. for intramuscular, subcutaneous, intravenous, epidural, intra-articular and intrathecal administration); or pharmaceutical compositions suitable for use in the form of ointments, lotions, creams, gels, patches, sublingual strips or films for parenteral (e.g., topical, buccal, sublingual, vaginal) administration and in unit dosage form. It can be. In one embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for topical (e.g., transdermal) delivery. Suitable transdermal delivery systems will be familiar to those skilled in the art, examples of which are described in Prausnitz and Langer (2008; Nature Biotechnol. 26(11):1261-1268), the contents of which are incorporated herein by reference. In other embodiments, the peptides as described herein and pharmaceutically acceptable salts thereof are formulated for sublingual or buccal delivery. Suitable sublingual and buccal delivery systems will be familiar to those skilled in the art, examples of which include dissolvable strips or films as described by Bala et al. (2013; Int. J. Pharm. Investig. 3(2):67-76) This is included, the contents of which are incorporated herein by reference.

Suitable pharmaceutical compositions and unit dosage forms thereof may contain conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any dosage proportional to the intended daily dosage range for use. It may contain a suitable effective amount of the active ingredient. Peptides and pharmaceutically acceptable salts thereof as described herein can be formulated for administration in a wide variety of enteral, topical and/or parenteral dosage forms. Suitable dosage forms may comprise a combination of two or more of the peptides described herein or pharmaceutically acceptable salts thereof as active ingredients.

In one embodiment, the composition is formulated for oral administration to humans. In another embodiment, the composition is formulated for oral administration to non-human subjects. In another embodiment, the composition is formulated for oral administration to a non-human subject selected from the group consisting of felines, canines, and equines.

In another embodiment, the composition is formulated for parenteral administration to humans. In another embodiment, the composition is formulated for parenteral administration to non-human subjects. In another embodiment, the composition is formulated for parenteral administration to non-human subjects selected from the group consisting of felines, canines, and equines. In one embodiment, parenteral administration is subcutaneous administration.

In another embodiment, the composition is formulated for topical administration to humans. In another embodiment, the composition is formulated for topical administration to non-human subjects. In another embodiment, the composition is formulated for topical administration to a non-human subject selected from the group consisting of felines, canines, and equines. In one embodiment, topical administration is transdermal administration.

In another embodiment, the composition is formulated for administration to humans by inhalation or insufflation. In another embodiment, the composition is formulated for administration to a non-human subject by inhalation or insufflation. In another embodiment, the composition is formulated for administration by inhalation or insufflation to a non-human subject selected from the group consisting of cats, dogs, and horses.

In another embodiment, the composition is formulated in a controlled release dosage form for administration to humans. In another embodiment, the composition is formulated in a controlled release dosage form to be administered to a non-human subject. In another embodiment, the composition is formulated in a controlled release dosage form to be administered to a non-human subject selected from the group consisting of felines, canines, and equines. Illustrative examples of suitable controlled release dosage forms are described elsewhere herein.

For preparing the pharmaceutical compositions described herein, pharmaceutically acceptable carriers can be solid or liquid. Illustrative examples of solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that can also act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material. In powders, the carrier may be a finely divided solid that exists in mixture with the finely divided active ingredient. In tablets, the active ingredient is mixed in appropriate proportions with a carrier having the necessary binding capacity and compressed into the desired shape and size.

In some embodiments, the powders and tablets contain from 5% or 10% to about 70% active compound. Illustrative examples of suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter, etc. . The term “preparation” is intended to include preparations of an active compound, with or without a carrier, together with an encapsulating material that provides a capsule in which the active ingredient is surrounded by a carrier. Similarly, cachets and lozenges are also contemplated herein. Solid forms suitable for oral administration include tablets, powders, capsules, pills, cachets, lozenges, etc.

When manufacturing suppositories, a low melting point wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and then stirred to uniformly disperse the active ingredients. Next, the molten homogeneous mixture is poured into a mold of an appropriate size, cooled, and solidified.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, a carrier known in the art to be suitable.

Liquid preparations include solutions, suspensions, and emulsions (e.g., water or water-propylene glycol solution). For example, parenteral injection liquid formulations can be formulated as solutions in aqueous polyethylene glycol solution.

The peptides and pharmaceutically acceptable salts thereof as described herein can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be administered in ampoules, prefilled syringes, or small volumes. It may be provided in unit dose form for injection or in multi-dose containers with added preservatives. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizing agents and/or dispersing agents. Alternatively, the active compound(s) may be in powder form obtained by aseptic isolation of a sterile solid or lyophilization from solution for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

Aqueous solutions suitable for oral use may be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers and thickening agents as desired.

Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with a viscous substance such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents.

Also contemplated herein are solid form formulations intended to be converted to liquid form formulations for oral administration immediately prior to use. These liquid forms include solutions, suspensions, and emulsions. In addition to the active ingredients, these preparations may contain colorants, flavoring agents, stabilizers, buffering agents, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

For topical administration to the epidermis, the peptides described herein and pharmaceutically acceptable salts thereof may be formulated as ointments, creams or lotions or as transdermal patches. Ointments and creams may be formulated, for example, with an aqueous or oily base by adding suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and usually also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

Preparations suitable for topical administration in the mouth include lozenges containing the active ingredients in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing active ingredients in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The solution or suspension is applied directly to the nasal cavity by conventional methods such as dropper, pipette or spray. The preparation may be provided in single or multiple dosage form. In the latter case of a dropper or pipette, this can be achieved by administering an appropriate, predetermined amount of solution or suspension to the patient. In the case of sprays, this can be achieved using a metered spray pump or inhaler. To improve nasal delivery and retention, the peptides used in the present invention can be encapsulated in cyclodextrins or formulated with agents expected to improve delivery and retention in the nasal mucosa.

Administration to the airway can also be achieved by providing the active ingredient in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane. This can be achieved by aerosol formulations, carbon dioxide or other suitable gases. The aerosol may conveniently contain a surfactant such as lecithin. The dosage of the drug can be adjusted by providing a metering valve.

Alternatively or additionally, the active ingredient may be in the form of a dry powder, for example a powder mixture of the compounds in a suitable powder base such as lactose, starch, hydroxypropylmethyl cellulose and starch derivatives such as polyvinylpyrrolidone (PVP). can be provided. Conveniently the powder carrier forms a gel within the nasal cavity. Powder compositions may be presented in unit dosage form, for example in capsules or cartridges of gelatin, or in blister packs from which the powder may be administered by inhaler.

In formulations for administration to the respiratory tract, including intranasal formulations, the peptide will generally have a small particle size, for example on the order of 1 to 10 microns or less. Such particle sizes can be obtained by means known in the art, such as micronization.

If desired, formulations tailored to provide controlled or sustained release of the active ingredient may be used as described elsewhere herein.

In one embodiment, the pharmaceutical formulation as described herein is preferably in unit dosage form. In this form the preparation is subdivided into unit doses containing the appropriate amount of active ingredient. Unit dosage forms can be packaged preparations, such as packaging containing individual quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Additionally, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or may be an appropriate number of these packaged forms.

In one embodiment, the compositions disclosed herein are formulated for oral administration to humans. In another embodiment, the compositions disclosed herein are formulated for oral administration to non-humans. In a further embodiment, the compositions disclosed herein are formulated for oral administration to non-humans selected from the group consisting of felines, canines, and equines.

In one embodiment, the compositions disclosed herein are formulated for administration to humans by inhalation or insufflation. In another embodiment, the compositions disclosed herein are formulated for administration to non-humans by inhalation or insufflation. In a further embodiment, the compositions disclosed herein are formulated for administration by inhalation or insufflation to a non-human selected from the group consisting of felines, canines, and equines.

In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for oral administration to human subjects. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for oral administration to non-human subjects. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for oral administration to non-human subjects selected from the group consisting of felines, canines, and equines.

In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for topical administration to human subjects. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for topical administration to non-human subjects. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for topical administration to non-human subjects selected from the group consisting of felines, canines, and equines. In one embodiment, topical administration is transdermal administration.

In other embodiments, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for administration to a human subject by inhalation or insufflation. In another embodiment, the peptides as described herein and pharmaceutically acceptable salts thereof are formulated for administration to non-human subjects by inhalation or insufflation. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for administration by inhalation or insufflation to non-human subjects selected from the group consisting of cats, dogs, and horses.

In another embodiment, the peptides as described herein and pharmaceutically acceptable salts thereof are formulated for administration to a human subject as a controlled release dosage form. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for administration to non-human subjects in a controlled release dosage form. In another embodiment, the peptides and pharmaceutically acceptable salts thereof as described herein are formulated for administration to a non-human subject as a controlled release dosage form, wherein the non-human subject is from the group consisting of felines, dogs and equines. is selected. In one embodiment, the controlled release dosage form is formulated for parenteral administration.

As noted elsewhere herein, multiple (i.e., multiple) divided doses may be administered at daily, weekly, monthly, or other suitable time intervals, or the doses may be reduced proportionally depending on the urgency of the situation. When multiple administration procedures are necessary or otherwise desirable, the compositions disclosed herein may be suitably formulated for administration via such multiple routes. For example, a primary dose may be administered parenterally (e.g., intramuscularly, intravenously, subcutaneously, etc.) to induce a rapid or otherwise acute therapeutic effect in a subject, followed by administration of the active agent over an extended period of time beyond the acute phase of treatment. It may be desirable to administer parenterally (e.g., enterally and/or topically) or in subsequent (e.g., second, third, fourth, fifth, etc.) doses to provide continued availability of the drug. You can. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for parenteral administration to an individual as a primary dose (i.e., as a parenteral dosage form) and are administered non-parenterally to an individual following the primary dose. Formulated for use (e.g., enteral and/or topical dosage forms). In one embodiment, the parenteral administration is selected from the group consisting of intramuscular, subcutaneous and intravenous. In a further embodiment, the parenteral administration is subcutaneous administration.

In another embodiment, the enteral administration is oral administration. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for parenteral administration to a subject as a first dose and for oral administration to a subject following the first dose (i.e., oral dosage form). .

In another embodiment, the enteral administration is topical administration. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for parenteral administration to a subject as a first dose, and for topical administration to a subject following the first dose (i.e., as an oral dosage form). . In one embodiment, topical administration is transdermal administration.

In other embodiments, the primary dose is administered parenterally (e.g., intramuscularly, intravenously, subcutaneously, etc.) to induce a rapid or otherwise acute therapeutic effect in the subject, and then administered over an extended period of time beyond the acute phase of treatment. It may be desirable to administer in subsequent (e.g., second, third, fourth, fifth, etc.) doses to provide controlled release of the active agent. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for oral administration to a subject for a first dose and are formulated for administration to the subject in controlled release form after the first dose. In one embodiment, the controlled release dosage form is formulated for parenteral administration.

Additionally, after the first dose is administered enterally (e.g., orally or rectally), subsequent doses (e.g., second, third, fourth, fifth, etc.) are administered topically (e.g., transdermally). ) It may be desirable to administer. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for enteral administration to a subject in a primary dose (i.e., enteral dosage form, oral or rectal) and, following administration of the primary dose, for topical administration to a subject. Formulated (e.g., transdermal or transmucosal dosage forms). In other embodiments, the peptides and compositions disclosed herein are formulated for topical administration selected from the group consisting of transdermal and transmucosal administration. In additional embodiments, the peptides and compositions disclosed herein are formulated for transdermal administration.

In another embodiment, a peptide or composition as disclosed herein is administered enterically (e.g., orally or rectally) as a first dose, followed by subsequent doses (e.g., second, third, It may be desirable to administer in a controlled release dosage form as described elsewhere herein (4th, 5th, etc.). Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for administration as an enteral primary dose and are formulated for administration in a controlled release dosage form, wherein the controlled release dosage form is for administration following the primary dose. It is formulated. In one embodiment, the enteral dosage is formulated for oral administration. In another embodiment, the controlled release dosage form is formulated for parenteral administration.

In one embodiment, a peptide or composition disclosed herein is administered topically (e.g., orally or rectally) as a first dose, followed by subsequent doses (e.g., second, third, fourth). , 5th, etc.), it may be desirable to administer in a controlled release dosage form as described elsewhere herein. Accordingly, in one embodiment, the peptides and compositions disclosed herein are formulated for topical administration as a primary dose and for administration in a controlled release dosage form, wherein the controlled release dosage form is for administration following the primary topical administration. It is formulated. In one embodiment, the topical dose is formulated for transdermal administration. In another embodiment, the controlled release dosage form is formulated for parenteral administration.

The invention will now be illustrated with reference to the following examples, which illustrate some preferred aspects of the invention. However, it should be understood that the specificity of the following description of the invention does not replace the generality of the previous description of the invention.

Example

Example 1: LanCL binding assay

Gel-based analysis of cross-linked proteins

Resuspend the dried pellet of LANCL1 previously photolabeled with a photoprobe in 30 μL SDS loading buffer (Bio Rad's XT sample buffer containing 2.5% v/v 2-mercaptoethanol) in the presence of other peptides or PBS/DMSO vehicle. and heated (60°C, 30 minutes). Proteins were analyzed using SDS-PAGE (4-15% CriterionTM TGX Stain-FreeTM Protein Gel, Bio Rad) and a ChemiDocTM MP Imaging System (Bio Rad) using green LED light as an excitation source and BP600/20nm emission filter. analyzed by in-gel fluorescence scanning. After fluorescence scanning in the gel, the gel was stained with Coomassie blue such that equal amounts of protein samples were loaded into each lane and imaged with the ChemiDocTM MP imaging system. Optical integration of each photoprobe in LANCL1 was performed by measuring the fluorescence intensity of the corresponding gel band using Image lab software (Bio Rad) and normalizing this value to the intensity value of the Coomassie blue-stained LANCL1 gel band to determine the loading difference. It was evaluated quantitatively by controlling.

result

As shown in Table 2, the peptide was found to bind specifically to LanCL1 and displace the known LanCL1 ligand, PAL-CRSVEGSCGF (SEQ ID NO: 22) from recombinant LanCL1 (rLanCL1). ED ₅₀ displacement values are listed in Table 2. Note that the cyclized peptide of SEQ ID NO:38 has an unexpectedly greater binding affinity for rLanCL1 when compared to its linear counterpart, SEQ ID NO:37. Similarly, the cyclized peptide of SEQ ID NO: 40 had better binding affinity for rLanCL1 compared to its linear counterpart, SEQ ID NO: 39.

peptide sequence
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number Substitution of SEQ ID NO: 22 from rLANCL1
~ED ₅₀ displacement value YLRIVQ C RSVEGS C GF One ~50-100μM C RSVEGS C G 3 ~150μM C RSRPVESS C S 14 ~100μM C RIIHNNN C 24 ~100-200μM QEQLERALNSS 37 >100-200μM C QEQLERALNSS C 38 ~50-100μM RALNSS 39 >200μM C RALNSS C 40 >50-100μM

Example 2: Respiratory Epithelial Cell Viability

By interacting with LANCL1, the peptide described in this patent appears to play a role in protecting cells from the damaging effects of chemical or oxidative stress. An assay was developed that stresses cells with a dose of Taxol, a chemotherapy agent that inhibits cell viability by 50% compared to untreated cells. The peptide was then added to the cell culture at increasing concentrations to assess its ability to restore the viability of Taxol-treated cells.

Briefly, A549 cells were cultured in culture medium (DMEM medium ref 11960-044 Thermoscientific, +10% FBS ref 10270-106 Gibco, Thermoscientific, +1% Na pyruvate ref S8636-100ML, Sigma, +1%, Glutamax ref35050061, Thermoscientific, +1% Penicillin-Streptomycin, ref 11074440001, Sigma) at 50000 A549 cells/well in opaque-walled multiwell plates. Background luminescence values were obtained using control wells containing cell-free medium. Cells were cultured overnight at 37°C and 5% CO ₂ .

Taxol (T7402-5 MG, Sigma-Aldrich) was added as a 10mM solution in DMSO to each well to a final concentration of 350uM, which resulted in 50% inhibition of proliferation compared to vehicle alone. 100 μL of medium + DMSO + peptide or medium + Taxol + peptide (various concentrations) was added to each well and incubated at 37°C, 5% CO ₂ for 16 hours.

Cell morphology, viability and confluency were assessed by phase contrast microscopy. They were then metabolically assayed using the CellTiter-Glo® Luminescent Cell Viability Assay (G7571, Promega - a homogeneous method to determine the number of viable cells in culture based on quantification of ATP present) according to the manufacturer's instructions. The number of active cells was quantified. A 100 μl volume of CellTiter-Glo® reagent was added to the 100 μl volume of cell culture medium present in each well. Cell lysis was induced by mixing the contents for 2 minutes on an orbital shaker, and then the plate was stabilized by incubating for 10 minutes at room temperature, and the luminescence signal was recorded using a CLARIOstar multi-well luminometer (BMG Labtech) with an integration time of 0.5 s.

result

As shown in Table 3, the peptide was found to restore the viability of A549 cells treated with a dose of Taxol resulting in a 50% reduction in proliferation compared to untreated cells in vitro. Consistent with the LanCL1 binding data in Table 2 above, the cyclized peptide of SEQ ID NO:38 was found to restore A549 viability, whereas its linear counterpart, SEQ ID NO:37, did not. Similarly, the cyclized peptide of SEQ ID NO: 10 was found to restore A549 viability, whereas its linear counterpart, SEQ ID NO: 11, did not. Unexpectedly, relatively short peptides of 3-, 4-, 5-, and 6-amino acid length (SEQ ID NOs: 39, 42, and 59-61) were also found to partially restore A549 viability. The peptide of SEQ ID NO: 40 (cyclized variant of SEQ ID NO: 39) also restored A549 viability. The peptide of SEQ ID NO: 9 (linear fragment of SEQ ID NO: 1) also restored the loss of cell viability induced by Taxol (see Figure 2).

To assess whether the effect of the peptide was dependent on LanCL expression, A549 cells were treated with LanCL1 siRNA (100 nM) for 48 h to knock down LanCL1 expression. Cells were then incubated in the presence of Taxol (IC50 ~350 μM), vehicle alone (dimethylsulfoxide; DMSO), or peptide of SEQ ID NO: 1 (diluted in DMSO) at concentrations of 1, 5, 25, 50, and 100 μM. Transfection with control siRNA (SiCTL) or siRNA against LanCL1 (SiLanCL1) did not alter A549 cell viability. As shown in Figure 1, the peptide of SEQ ID NO: 1 had no significant effect on the viability of untransfected A549 cells (NT) or A549 cells transfected with SiCTL in the absence of Taxol. In SiLanCL1-transfected cells, the peptide of SEQ ID NO: 1 inhibited A549 proliferation at higher doses.

In the presence of 350 μM Taxol, the presence of the peptide of SEQ ID NO: 1 rescued the loss of viability of untransfected A549 cells (NT) or A549 cells transfected with SiCTL. This effect represents a protective effect on epithelial cells. In contrast, the peptide of SEQ ID NO: 1 did not rescue the negative effect of Taxol on A549 viability.

These data show that peptides containing the amino acid sequence of formula (I) can rescue the negative effects of Taxol-induced stress on epithelial cell viability and that this rescue effect is dependent on LanCL1.

peptide sequence
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number Expected activity in A549 proliferation + stress assay YLRIVQ C RSVEGS C GF One strong C SGRVSE C GF (variant in the scrambled loop of SEQ ID NO: 2) 8 inactive RSVEGS 9 strong S C RSRPVESS C 10 strong SCRSRPVESSC (linear variant of SEQ ID NO: 10) 11 inactive C RSRPVESS C S 14 strong C RIIHNNN C 24 strong QEQLERALNSS 37 inactive C QEQLERALNSS C 38 middle RALNSS 39 weakness C RALNSS C 40 strong KALPRS 42 strong RALNS 59 Weak-medium RALN 60 weakness RAL 61 weakness ALNSS 63 middle

Active Score:

Inactive - no activity up to 100 μM;

Mild - partial or variable recovery of cell viability at 50-100 μM;

Medium - Moderate dose-dependent recovery of cell viability at >25 μM;

Strong - clear dose-dependent recovery of cell viability above 1-5 μM

Example 3: Mouse model of influenza A infection

Six- to eight-week-old C57BL/6 male mice were maintained in a specific pathogen-free physical containment level 2 (PC2) animal research facility at Monash Medical Center. All experimental procedures were approved by the Hudson Animal Ethics Committee, and experimental procedures were performed in accordance with approved guidelines. The IAV strain used in this study was HKx31(H3N2), a high-yield reassortant of A/PR/8/34(H1N1) carrying the surface glycoprotein of A/Aichi/2/1968(H3N2). HKx31 was grown in 10-day hatched eggs and titrated in Madin-Darby Canine Kidney (MDCK) cells according to standard procedures.

For viral infection studies, groups of eight male C57BL/6 mice were randomly assigned. Mice were lightly anesthetized and infected intranasally with 10 ⁵ PFU of HKx31(H3N2) in 50 μl PBS (previously shown to cause severe disease (Rosli et al., 2019; Tate et al., 2016)). Mice were treated with the peptides described herein (5 or 20 mg/kg as indicated) via the intranasal route at the indicated time points. Control mice were treated with PBS alone. Mice were weighed daily and assessed for visual signs of clinical disease, including inactivity, ruffled fur, respiratory distress, and huddled behavior. Animals that lost ≥20% of their original body weight or showed severe clinical signs of disease were euthanized. Bronchoalveolar lavage (BAL) was obtained immediately after euthanasia by washing the lungs three times with 1 mL of PBS. The lungs were then removed and immediately frozen in liquid nitrogen. The titer of infectious virus in lung homogenates was determined by standard plaque assay on MDCK cells.

Cytokine quantification in mouse BAL fluid and serum.

To detect cytokines, BAL fluid was collected and stored at -80°C. The levels of IL-6, MCP-1/CCL2, IFNγ, IL-10, IL-12p70, and TNFα proteins were determined by cytokine bead array (CBA) using a mouse inflammation kit (Becton Dickinson). The levels of mouse IFNα were determined by sandwich ELISA using mouse monoclonal clone F18 (Thermo Scientific) and rabbit polyclonal antibody (PBL) (Thomas et al., 2014). Levels of mouse IFNβ were determined by sandwich ELISA using mouse monoclonal clone 7F-D3 (Abcam) and rabbit polyclonal antibody (PBL) (Thomas et al., 2014). Mouse IFNλ _2/3 was quantified by ELISA (R&D Systems).

Recovery and characterization of mouse leukocytes

For flow cytometry, BAL cells were treated with red blood cell lysis buffer (Sigma Aldrich) and cell number and viability were assessed by trypan blue exclusion using a hemocytometer. BAL cells were incubated with Fc block (2.4G2; eBiosciences) and then stained with fluorochrome-conjugated monoclonal antibodies against Ly6C, Ly6G, CD11c, and IA ^b (MHC-II) (BD Biosciences, USA). Neutrophils (Ly6G ⁺ ), macrophages (CD11c ⁺ IA ^blow ), dendritic cells (DC; CD11c ⁺ IA ^bhigh ), and inflammatory macrophages (Ly6G ^- Ly6C ⁺ ) were quantified by flow cytometry as previously described (Rosli et al. al., 2019; Tate et al., 2016). Live cells (propidium iodide negative) were analyzed using a BD FACS Canto II flow cytometer (BD Biosciences) and FlowJo software (BD Biosciences).

Assessment of pulmonary edema and vascular leakage

The ratio of wet weight to dry weight of the lung was used as an indicator of fluid accumulation in the lung. After the mice were euthanized, the lungs were surgically excised, dried blotted, and immediately weighed (wet weight). The lung tissue was then dried in an oven at 55°C for 72 hours and reweighed to dry weight. To assess tissue swelling, the ratio of wet weight to dry weight was calculated for each animal (Tate et al., 2009; Tate et al., 2010). The protein concentration of cell-free BAL supernatant was measured by adding Bradford protein dye (Tate et al., 2009; Tate et al., 2010). A standard curve was prepared using bovine serum albumin and the optical density (OD) at 595 nm was determined.

result

As shown in Table 4, treatment with the cyclic peptide of SEQ ID NO: 1 (10 mg/kg single dose) increased the infiltration of polymorphonuclear cells (PMN), viral titers, and IL-6 levels in bronchial lavage fluid induced by viral infection. decreases routinely. At a single 10 mg/kg dose, the peptides of SEQ ID NOs: 1, 2, 9, 29, 37-39, 42, 56, and 59-61 were as effective as the peptide of SEQ ID NO: 38 in reducing PMN infiltrates in BAL fluid, whereas the peptides of SEQ ID NOs: Peptides numbered 23, 40, 41, 50, 57 and 58 were relatively less effective than peptide numbered 38 in reducing PMN infiltrates in BAL fluid at a single 10 mg/kg. Treatment with any of the peptides of SEQ ID NOs: 43, 44, 47-49, and 52 showed no changes in PMN infiltrates in BAL fluid at a single 10 mg/kg dose (activity score: 0 = inactive; 1 = SEQ ID NO less active than the peptide of SEQ ID NO:38; 2 = similar to or more active than the peptide of SEQ ID NO:38).

Compared to SEQ ID NO: 38, when treated with any of the peptides of SEQ ID NO: 1, 9, 23, 29, 37, 38, 42, 50, 52, 56, and 59-61 (10 mg/kg single dose) on day 3 It has been shown to be effective in reducing virus titer.

Cytokine profiles were variable in lung and serum samples, but data were treated with any of the peptides SEQ ID NOs: 1, 9, 23, 29, 37, 38, 42, 44, 47, 49, 50, 52, 56, and 59-61. One was shown to be effective in reducing IL-6 levels in BAL fluid to levels comparable to the peptide of SEQ ID NO:38.

amino acid sequence
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number PMN activity score Virus Titer Activity Score BAL IL-6 activity score YLRIVQ C RSVEGS C GF One 2 2 2 C RSVEGS C GF 2 2 RSVEGS 9 2 2 2 RPVESS 23 One 2 One RIHNN 29 2 2 2 QEQLERALNSS 37 2 2 2 C QEQLERALNSS C 38 2 2 2 RALNSS 39 2 2 2 C RALNSS C 40 One 0 0 RALNSS (cyclization; R1 conjugated to S6) 41 One KALPRS 42 2 2 2 HIVESS 44 0 One 2 RAVESS 47 0 One 2 RALNST 49 0 0 2 RALQSS 50 One 2 2 PALNTS 52 0 2 2 RSVEG 56 2 2 2 RSVE 57 One 0 0 R.S.V. 58 One 0 0 RALNS 59 2 2 2 RALN 60 2 2 2 RAL 61 2 2 2 ALNSS 63 0 LNs 64 0

Example 4: In vivo model of neuropathic pain

This study was conducted to evaluate the analgesic effect of the peptides described herein on neuropathic pain in vivo using a model of nerve contraction in Chung rats. Briefly, adult male Sprague-Dawley rats, 8-9 weeks old and weighing 220-250 g at the time of surgery, were purchased from Charles River UK Ltd.

The animals were housed in groups of four in an air-conditioned room with a 12-hour light/dark cycle. Food and water were freely available. They were allowed to acclimate to the experimental environment for 3 days by placing them on a raised metal mesh for at least 40 minutes. Baseline paw withdrawal threshold (PWT) was tested using serial graduated von Frey hairs on 3 consecutive days before surgery and reassessed before drug administration on days 6–8 and 12–14 after surgery.

Each rat was anesthetized with 5% isoflurane mixed with oxygen (2 L/min) and then injected intramuscularly (i.m.) with 90 mg/kg of ketamine and 10 mg/kg of xylazine. The back was shaved and disinfected with povidone iodine. The animal was placed in the prone position and a para-medial incision was made in the skin covering the L4-6 level. The L5 spinal nerve was carefully isolated and tightly ligated with 6/0 silk suture. Then, after complete hemostasis, the wound was closed in layers. A single dose of antibiotic (Amoxipen, 15 mg/rat, i.p.) was administered regularly to prevent postoperative infection. The animals were kept in a temperature-controlled recovery room until fully awake and then returned to their home cages.

The vehicle (1% DMSO in PBS) or peptide was administered intramuscularly (i.m.) to the leg contralateral to the injury site. Administration was performed by a second experimenter. Rats with a proven neuropathic pain condition were randomly divided into five experimental groups: 1 ml/kg vehicle, 0.1, 0.5, 1 and 5 mg/kg peptide.

There were 8 animals in each group. Animals were placed in individual Perspex boxes over raised metal mesh for at least 40 minutes before testing. Starting with the filament of the weakest force (approximately 1 g), each filament was applied vertically to the center of the ventral surface of the paw until slightly bent for 6 s. If the animal withdrew or lifted its paw upon stimulation, a hair with a lower force than that tested was immediately used. If no response was observed, hair with a stronger force was immediately tested. The minimum amount of force required to induce a reliable response (positive in 3 out of 5 trials) was recorded as the PWT value.

Drug testing was performed 12 to 14 days after surgery. PWT was assessed before, 1, 2, and 4 hours after drug or vehicle administration. Between two adjacent testing time points, animals were returned to their home cages (approximately 30–60 min) to rest. Peptides were administered by a single intramuscular injection (IM) into the ipsilateral limb at a dose of about 0.1 mg/kg body weight to about 5 mg/kg body weight.

result

As shown in Table 5 below, the peptides of SEQ ID NOs: 1, 2, 3, 10, 24, and 37, including the 6-mer peptide of SEQ ID NO: 39, are administered orally, subcutaneously, and/or intramuscularly (2-10 mg/day). Oral doses in the kg range, subcutaneous doses in the range of 0.1-3 mg/kg, and intramuscular doses in the range of 0.5-5 mg/kg) reduced neuropathic pain in the Chung model.

amino acid sequence
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number In Vivo Active Chung Model of Neuropathic Pain YLRIVQ C RSVEGS C GF One +++ Oral, SC and IM administration C RSVEGS C GF 2 +++ Oral and IM administration C RSVEGS C G 3 +++ Oral and IM administration; S C RSRPVESS C 10 +++ Oral and IM administration C RIIHNNN C 24 +++ Oral and IM administration QEQLERALNSS 37 ++ SC administration RALNSS 39 +++ SC administration

Example 5: In vivo model of systemic encephalomyocarditis virus (EMCV) infection

In preliminary experiments using a mouse model of systemic encephalomyocarditis virus (EMCV) infection, a decrease in the number of intraperitoneal neutrophils and inflammatory macrophages was observed after intraperitoneal administration of peptides of SEQ ID NOs: 1, 37, and 38 (Table 6). These observations were associated with a decrease in circulating MCP-1, a cytokine that promotes the migration and activation of both immune cells.

amino acid sequence
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number Neutrophil infiltration in the peritoneum YLRIVQ C RSVEGS C GF One good active QEQLERALNSS 37 medium active C QEQLERALNSS C 38 good active

Example 6: In vivo model of neuropathic pain (II)

The spinal nerve ligation (Chung) model was prepared as described in Example 4 above. Briefly, 64 adult male Sprague-Dawley rats, aged 8–9 weeks and weighing 250–350 g at the time of surgery, were purchased from Charles River UK Ltd. The animals were housed in groups of four in an air-conditioned room with a 12-hour light/dark cycle. Food and water were freely available. They were allowed to acclimate to the experimental environment for 3 days by placing them on a raised metal mesh for at least 40 minutes. Baseline paw withdrawal threshold (PWT) was tested using serial graduated von Frey hairs on 3 consecutive days before surgery and reassessed before drug administration on days 6–8 and 12–14 after surgery.

Each rat was anesthetized with 5% isoflurane mixed with oxygen (2 L/min) and then injected intramuscularly (i.m.) with 60 mg/kg of ketamine and 10 mg/kg of xylazine. The back was shaved and disinfected with povidone iodine. The animal was placed in the prone position and a para-medial incision was made in the skin covering the L4-6 level. The L5 spinal nerve was carefully isolated and tightly ligated with 6/0 silk suture. Then, after complete hemostasis, the wound was closed in layers. A single dose of antibiotic (Amoxipen, 15 mg/rat, i.p.) was administered regularly to prevent postoperative infection. The animals were kept in a temperature-controlled recovery room until fully awake and then returned to their home cages.

Animals with proven neuropathic pain conditions were randomly divided into four experimental groups: vehicle (initially 5% DMSO, then 0.9% saline), 3 mg/kg LAT9997, 3 mg/kg LAT9997 x1, and 3 mg/kg LAT9997 x1. kg LAT1233x1. Each group included 6 animals.

RSVEGS (SEQ ID NO: 9; LAT9997), SVEGS (SEQ ID NO: 62; LAT9997x1) and ALNSS (SEQ ID NO: 63; LAT1233x1) were first dissolved in 5% DMSO and then in 0.9% saline, which also served as vehicle control. It has been done. All compounds were provided by GenScript for Lateral Pharma. All vehicles/compounds were administered intravenously at 1 ml/kg body weight.

Paw withdrawal threshold (PWT)

The animals were placed in individual perspex boxes over raised metal mesh for a minimum of 40 minutes. Starting with the filament of the weakest force (approximately 1 g), each vFH filament was applied vertically to the center of the ventral surface of the paw until slightly bent for 6 s. If the animal withdrew or raised its paw upon stimulation, a filament with a lower force than that tested was immediately used. If no response was observed, a filament with a stronger force was immediately tested. The minimum amount of force required to induce a reliable response (positive in 2 out of 3 trials) was recorded as the PWT value.

PWT was assessed once daily for 3 days before surgery (pre D1, Pre D2, and D0) and on day 7 after surgery to monitor the occurrence of mechanical noxious stimulation pain.

All drug tests were performed 13 to 17 days after surgery. PWT was assessed before (BL) and 1 and 2 hours after drug or vehicle administration.

One-way analysis of variance (ANOVA) (IBM Statistics SPSS, version 27) was used for statistical analysis to compare PWT of different groups at the same time point. Where appropriate, Fisher's least significant difference (LSD) post-hoc test was used to compare drug treatment groups with controls. Values at different time points within the same group were compared using paired Student's t-test (Microsoft Excel 365). To characterize drug-induced changes in PWT relative to vehicle, vehicle values were subtracted from the appropriate drug values. The level of significance was set at P<0.05.

result

PWT ranged from 10.0 to 15.0 g in naïve rats (prior to surgery). The average PWT was 14.17 ± 0.83 g and 15.00 ± 0.00 g for the ipsilateral (left) and contralateral (right) hindpaws in the vehicle group the day before surgery, respectively. The average PWT in the LAT9997 group was 15.00 ± 0.00 g in both left and right hind paws, and in the LAT9997 x 1 and LAT1233 x 1 groups it was 15.00 ± 0.00 g in both left and right hind paws. There was no statistically significant difference between groups (P > 0.05, one-way analysis of variance).

At 7 days after surgery, the ipsilateral PWT of the ligated nerve was significantly lower than that determined preoperatively (6.00±0.52 g for the vehicle group, 5.67±0.33 g for the LAT9997 group, and 6.33±0.33 g for the LAT9997 x 1 group; 5.33 ± 0.42 g for the LAT1233 x 1 group; P < 0.001 for all groups compared to preoperative values, paired Student's t-test). PWT on the contralateral side was not significantly affected by surgery (14.17 ± 0.83 g for the LAT1233 x 1 group and 15.00 ± 0.00 g for all other groups, P > 0.05 for all groups compared to preoperative values, paired Student's t-test).

Effect of vehicle (5% DMSO) on PWT

Before vehicle (5% DMSO) administration on test day, PWT of the (ipsilateral) hindpaw was significantly lower compared to the contralateral hindpaw: 3.33 ± 0.42 g on the ipsilateral side and 14.17 ± 0.83 g on the contralateral side (see Figures 3 and 4). After vehicle treatment, ipsilateral PWT was not significantly affected from 1 to 4 hours after administration, and the amounts were 3.67 ± 0.61 g, 3.67 ± 0.61 g, and 4.00 ± 0.89 g at 1, 2, and 4 hours, respectively ( All P > 0.05 compared to pre-dose levels, paired Student's t-test, see Figure 3 and Table 7). On the contralateral side, PWT remained unaffected (14.17 ± 0.83 g at all time points, see Figure 4 and Table 8).

Impact of LAT9997 on PWT

At 3 mg/kg, LAT9997 induced a significant increase in ipsilateral hindpaw PWT in Chung model rats (see Figure 3 and Table 7). The effect was significant from 1 hour after administration: 3.33 ± 0.42 g before administration compared to 7.83 ± 1.72 g at 1 hour after administration (P < 0.05, compared with pre-administration level, paired Student's t-test). At 2 hours after administration, PWT further increased to 9.67 ± 1.73 g (P < 0.01, compared to pre-administration level, paired Student's t-test). At 4 hours after administration, PWT decreased slightly to 8.17 ± 1.60 g (P < 0.05, compared to pre-administration level, paired Student's t-test). PWT was significantly different from that recorded in the vehicle group at 2 and 4 hours post-dose (both P < 0.05, one-way ANOVA).

The contralateral PWT did not change during the entire observation period (14.17 ± 0.83 g before administration and 15.00 ± 0.00 g at 1, 2, and 4 hours after administration). Contralateral PWT was not significantly different from PWT in the vehicle group at any time post-dose (P > 0.05, one-way ANOVA, see Figure 4 and Table 8).

Impact of LAT1233x1 on PWT

LAT1233x1 at 3 mg/kg also caused a rapid and significant increase in PWT of the ipsilateral hind paw of Chung model rats starting 1 hour after administration: 3.33 ± 0.42 g before administration compared to 10.67 ± 1.67 g at 1 hour after administration. P < 0.01, compared to pre-administration level, paired Student's t-test). At 2 hours after administration, PWT increased slightly further to 11.50 ± 1.80 g (P < 0.01, compared to pre-administration level, paired Student's t-test). At 4 hours after administration, PWT decreased slightly to 10.17 ± 1.17 g (P < 0.01, compared to pre-administration level, paired Student's t-test). At all time points post-dose, PWT was significantly different from that recorded in the vehicle group (all P < 0.01, one-way ANOVA; see Figure 3 and Table 7).

The PWT of the contralateral side did not change significantly during the entire observation period (15.00 ± 0.00 g before administration, 15.00 ± 0.00 g, 14.17 ± 0.83 g, and 15.00 ± 0.00 g at 1, 2, and 4 hours after administration, respectively). Contralateral PWT was not significantly different from PWT in the vehicle group at any time post-dose (P > 0.05, one-way ANOVA, see Figure 4 and Table 8).

Table 7: Ipsilateral PWT changes over time in Chung model rats following administration of LAT9997, LAT9997x1, and LAT1233x1.

Each value represents the mean (±1 SEM). PWT is expressed in g assessed using graduated von Frey hairs.

▲, ▲▲: P < 0.05 and 0.01, respectively, compared to pre-administration values (paired Student's t-test);

*, **, ***: P < 0.05, 0.01, and 0.001, respectively (one-way ANOVA) compared with vehicle group at the same time point.

Table 8: Contralateral PWT changes over time in Chung model rats following administration of LAT9997, LAT9997x1 and LAT1233x1.

Each value represents the mean (±1 SEM). PWT is expressed in g assessed using graduated von Frey hairs.

There were no statistically significant differences across time points in any treatment group (paired Student's t-test) and between groups at the same time point.

(One-way ANOVA). n = 6 for each group.

Table 9: Amino acid sequence

peptide
(Bold and underlined cysteine residues indicate cyclic peptides with disulfide bonds between the cysteine residues) sequence number YLRIVQ C RSVEGS C GF One C RSVEGS C GF 2 C RSVEGS C G 3 C RSVEGS C 4 C RRFVESS C AF 5 C RRFVESS C A 6 PAL- C RSVEGS C G 7 C SGRVSE C GF 8 RSVEGS 9 S C RSRPVESS C 10 SCRSRPVESSC (linear, acyclic variant of SEQ ID NO: 10) 11 C NVPSSSHEE C 12 C RSRPVESS C 13 C RSRPVESS C S 14 S C RSRPVESS C S 15 IDPSSEAPGHS C RSRPVESS C 16 C RSRPVESS C SSKFSWDEYEQYKKE 17 S C RARPVESS C 18 S C RSRPAESS C 19 S C RSRPVEAS C 20 S C RSRPVESA C 21 PAL- C RSRPVESS C S 22 RPVESS 23 C RIIHNNN C 24 C RIIHNNN C G 25 C RIVYDSN C 26 C RIVYDSN C G 27 PAL- C RIIHNNN C 28 RIHNN 29 C RSRFVKKDGH C 30 GGSRFVLSQQALSC 31 FQDRVEFSGNPSK 32 C RNFFWKTFSS C 33 C VSSP C 34 RRRRRRRR 35 C RRRRRRRR C 36 QEQLERALNSS 37 C QEQLERALNSS C 38 RALNSS 39 C RALNSS C 40 R ALNS S (cyclization; R1 conjugated to S6) 41 KALPRS 42 RALRTK 43 HIVESS 44 HLADTS 45 RIVETS 46 RAVESS 47 RALNSSdaa (variant of SEQ ID NO: 11 with D-serine at position 6) 48 RALNST 49 RALQSS 50 RALNTS 51 PALNTS 52 RAINSS 53 RALNTT 54 RALNQS 55 RSVEG 56 RSVE 57 R.S.V. 58 RALNS 59 RALN 60 RAL 61 SVEGS 62 ALNSS 63 LNs 64 EQLERALNSS 65

Claims (46)

A peptide capable of binding to a lanthionine synthase C-like (LanCL) protein, said peptide comprising an amino acid sequence of formula (I):
X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (I)
here:
(a) X ₁ is selected from the group consisting of lysine, arginine and histidine, or X ₁ is absent;
(b) X ₂ is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine, tyrosine and serine;
(c) X ₃ is selected from the group consisting of glycine, alanine, valine, leucine and isoleucine;
(d) _X4 is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, aspartic acid, lysine, glutamic acid, proline and histidine, or _X4 is absent;
(e) X ₅ is selected from the group consisting of serine, cysteine, threonine, asparagine, arginine, glutamine, tyrosine, lysine, histidine and glycine, or X ₅ is absent;
(f) X ₆ is selected from the group consisting of serine, cysteine, threonine, asparagine, glutamine, tyrosine, and histidine, or X ₆ is absent,
The peptide is 3 to 20 amino acids in length;
The amino acid sequence of the peptide does not include CRSRPVESSC, CRSVEGSCG, or CRIIHNNNC;
The peptide is not a linear peptide comprising the amino acid sequence EQLERALNSS. The peptide according to claim 1, wherein X ₁ is arginine. The peptide according to claim 1 or 2, wherein X ₂ is selected from the group consisting of alanine, isoleucine, proline, phenylalanine, and serine. The peptide according to any one of claims 1 to 3, wherein X ₃ is selected from the group consisting of valine, leucine and isoleucine. The peptide according to any one of claims 1 to 4, wherein X ₄ is selected from the group consisting of asparagine, glutamic acid, proline and histidine, or X ₄ is absent. The peptide according to claim 5, wherein X ₄ is selected from the group consisting of asparagine, glutamic acid, proline and histidine. 6. The peptide of claim 5, wherein X ₄ is absent. 8. The peptide according to any one of claims 1 to 7, wherein X ₅ is selected from the group consisting of serine, asparagine and glycine, or X ₅ is absent. The peptide of claim 8, wherein X ₅ is selected from the group consisting of serine, asparagine, and glycine. 9. The peptide of claim 8, wherein X ₅ is absent. 11. The peptide according to any one of claims 1 to 10, wherein X ₆ is serine or asparagine, or X ₆ is absent. The peptide according to claim 11, wherein X ₆ is serine or asparagine. 12. The peptide of claim 11, wherein X ₆ is absent. In claim 1,
(a) X ₁ is selected from the group consisting of lysine, arginine, and conservative amino acid substitutions of any of these;
(b) X ₂ is selected from the group consisting of alanine, isoleucine, proline, serine and conservative amino acid substitutions of any one of these;
(c) X ₃ is selected from the group consisting of valine, leucine, isoleucine and conservative amino acid substitutions of any one of these;
(d) X ₄ is selected from the group consisting of asparagine, glutamic acid and conservative amino acid substitutions of any of these, or X ₄ is absent;
(e) X ₅ is selected from the group consisting of serine, glutamine and conservative amino acid substitutions of any of these, or X ₅ is absent;
X ₆ is serine or a conservative amino acid substitution thereof, or X ₆ is absent. In claim 14,
(a) X ₁ is lysine or arginine;
(b) X ₂ is selected from the group consisting of alanine, isoleucine, proline and serine;
(c) X ₃ is selected from the group consisting of valine, leucine and isoleucine;
(d) X ₄ is asparagine or glutamic acid, or X ₄ is absent;
(e) X ₅ is serine or glutamine or X ₅ is absent;
(f) a peptide wherein X ₆ is serine or X ₆ is absent. 16. The method of any one of claims 1 to 15, wherein the peptide is selected from the group consisting of RAL, RALN, RALNS, RALNSS, RSV, RSVE, RSVEG, RSVEGS, RPV, RPVE, RPVES, RPVESS, RII, RIIH, RIIHN and RIIHNN. A peptide comprising a selected amino acid sequence. 16. The method of any one of claims 1 to 15, wherein the peptide is selected from the group consisting of RAL, RALN, RALNS, RALNSS, RSV, RSVE, RSVEG, RSVEGS, RPV, RPVE, RPVES, RPVESS, RII, RIIH, RIIHN and RIIHNN. A peptide consisting of a selected amino acid sequence. 16. The peptide according to any one of claims 1 to 15, wherein the peptide comprises the amino acid sequence RALNSS. The peptide according to any one of claims 1 to 15, wherein the peptide consists of the amino acid sequence RALNSS. The peptide according to any one of claims 1 to 19, wherein the peptide is a cyclic peptide. The peptide according to claim 20, wherein the cyclic peptide is formed by a disulfide bond between two cysteine residues. 22. The peptide of claim 21, wherein the cyclic peptide comprises the amino acid sequence CQEQLERALNSSC. The peptide according to claim 21, wherein the cyclic peptide consists of the amino acid sequence CQEQLERALNSSC. 22. The peptide of claim 21, wherein the peptide comprises the amino acid sequence CRALNSSC. The peptide according to claim 21, wherein the peptide consists of the amino acid sequence CRALNSSC. The peptide according to any one of claims 1 to 25, wherein the peptide is capable of competing with a peptide consisting of the amino acid sequence CRSVEGSCG for binding to LanCL. The peptide of claim 1 wherein X ₁ is absent. 16. The peptide of claim 14 or 15, wherein X ₁ is absent. The peptide according to claim 27 or 28, wherein the peptide comprises the amino acid sequence ALNSS. The peptide according to claim 27 or 28, wherein the peptide consists of the amino acid sequence ALNSS. 31. The peptide according to any one of claims 1 to 30, wherein at least one of the amino acids of formula (I) is a D-amino acid. 32. The peptide according to any one of claims 1 to 31, wherein the peptide is not a linear peptide comprising the amino acid sequence QEQLERALNSS. A pharmaceutical composition comprising the peptide of any one of claims 1 to 32. A method of treating a condition in an individual comprising administering a therapeutically effective amount of the peptide of any one of claims 1 to 32 to the individual in need thereof. The method of claim 34, wherein the condition is pain, inflammatory airway disease, microbial infection, respiratory tract infection, migraine, sarcopenia, impaired glucose tolerance, diabetes, obesity, metabolic disease and obesity-related conditions, osteoarthritis, muscle disorder, wasting disorder, aging, cachexia. , anorexia, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, neuromuscular junction disease, inflammatory myopathy, ophthalmic condition, central nervous system condition, neurodegenerative condition, Parkinson's disease, Alzheimer's disease, Burns, wounds, injuries or trauma, conditions associated with elevated LDL cholesterol, conditions associated with impaired chondrocyte, proteoglycan or collagen production or quality, conditions associated with impaired cartilage tissue formation or quality, impaired muscle, ligament or tendon mass, form or function. A method selected from the group consisting of conditions associated with, conditions associated with inflammation, trauma or genetic abnormalities affecting muscle or connective tissue, and bone disorders. The method of claim 35, wherein the condition is pain. 37. The method of claim 36, wherein the condition is neuropathic pain. 36. The method of claim 35, wherein the condition is an inflammatory airway disease. The method of claim 38, wherein the inflammatory airway disease is chronic obstructive pulmonary disease. 36. The method of claim 35, wherein the condition is a respiratory tract infection. 41. The method of claim 40, wherein the respiratory tract infection is a respiratory viral infection. 42. The method of claim 41, wherein the virus is an influenza virus or a coronavirus. Use of a therapeutically effective amount of the peptide of any one of claims 1 to 32 in the manufacture of a medicament for the treatment of a condition in a subject in need of such treatment. The method of claim 43, wherein the condition is pain, inflammatory airway disease, microbial infection, respiratory tract infection, migraine, sarcopenia, impaired glucose tolerance, diabetes, obesity, metabolic disease and obesity-related conditions, osteoarthritis, muscle disorder, wasting disorder, aging, cachexia. , anorexia, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, neuromuscular junction disease, inflammatory myopathy, ophthalmic condition, central nervous system condition, neurodegenerative condition, Parkinson's disease, Alzheimer's disease, Burns, wounds, injuries or trauma, conditions associated with elevated LDL cholesterol, conditions associated with impaired chondrocyte, proteoglycan or collagen production or quality, conditions associated with impaired cartilage tissue formation or quality, impaired muscle, ligament or tendon mass, form or function. Uses selected from the group consisting of conditions associated with, conditions associated with inflammation, trauma or genetic abnormalities affecting muscle or connective tissue, and bone disorders. 33. A peptide according to any one of claims 1 to 32 for use in treating a condition in an individual in need thereof. The method of claim 45, wherein the condition is pain, inflammatory airway disease, microbial infection, respiratory tract infection, migraine, sarcopenia, impaired glucose tolerance, diabetes, obesity, metabolic disease and obesity-related conditions, osteoarthritis, muscle disorder, wasting disorder, aging, cachexia. , anorexia, AIDS wasting syndrome, muscular dystrophy, neuromuscular disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, neuromuscular junction disease, inflammatory myopathy, ophthalmic condition, central nervous system condition, neurodegenerative condition, Parkinson's disease, Alzheimer's disease, Burns, wounds, injuries or trauma, conditions associated with elevated LDL cholesterol, conditions associated with impaired chondrocyte, proteoglycan or collagen production or quality, conditions associated with impaired cartilage tissue formation or quality, impaired muscle, ligament or tendon mass, form or function. A peptide selected from the group consisting of conditions associated with, conditions associated with inflammation, trauma or genetic abnormalities affecting muscle or connective tissue, and bone disorders.